Rab14 restricts pathogens by promoting V-ATPase lysosomal delivery to drive lysosomal acidification

Domain: chemistry
Published: 2026-03-02
Editorial outcome: major_revision

Abstract
Host restriction factors mediate intrinsic immunity against infections, thus serving as promising targets for host-directed therapy (HDT) against drug-resistant pathogens. While restriction factors counteracting viruses have been extensively studied, those targeting bacteria, particularly those with broad-spectrum activity, remain largely unexplored. Here, through screening for host factors promoting lysosomal acidification, a crucial process clearing pathogens, we identify the host small GTPase Rab14 as a restriction factor with broad-spectrum activity against multiple bacteria and viruses. Mechanistically, upon pathogen infections, GTP-bound Rab14 increases and binds to the calcium/calmodulin-dependent protein kinase type 2 delta (CAMK2D), suppressing CAMK2D-mediated phosphorylation of V0a1, the critical sub unit determining V-ATPase localization, thus promoting V0a1 binding to the COPⅡ complex to facilitate V-ATPase trafficking from the endoplasmic reticulum to lysosomes, resulting in lysosomal acidification and pathogen clearance. Taken together, our data demonstrate an unrecognized intrinsic immune mechanism mediated by Rab14-CAMK2D-V-ATPRase axis, which might be a promising target for infectious diseases.

Introduction
Infectious diseases continue to pose major global health challenges, exacerbated by the rise of drug-resistant pathogens that are projected to kill approximately 39 million people by 20501, 2. This problem arises because that traditional antibiotics or antivirals directly target pathogens, exerting lethal selective pressure that drives resistance mutations. Against this backdrop, host-directed therapy (HDT) has emerged as a transformative strategy that leverages host biological mechanisms to enhance pathogen clearance while minimizing resistance development3. Intrinsic immunity functions as the cell-autonomous first-line defense mechanism against pathogen infections, which is mediated by the constitutively expressed host restriction factors4, 5. In the past decade, studies have reported multiple host restriction factors (such as TRIM5, APOBEC3G, IFITMs) against virus infections through directly inhibiting viral replication or reprogramming host anti-viral immune responses6-8. However, restriction factors that counteract bacterial infections, as well as those with broad-spectrum activity against both viral and bacterial pathogens, remain poorly defined. In particular, their specific and dynamic regulatory mechanisms during infection remain elusive. This gap impedes the development of HDT strategies targeting bacterial and polymicrobial infections.

Lysosomes are membrane-bound organelles that serve as the primary degradative hubs in cells, playing a pivotal role in the cell-intrinsic defense against a wide range of pathogens9. Hydrolytic enzymes, such as cathepsins, are the main executors of lysosomal degradation functions, and their optimal activity is critically dependent on the maintenance of an acidic luminal pH within lysosomes, which is established and maintained by the vacuolar ATPase (V-ATPase), a proton pump that actively transports protons into the lysosomal lumen10, 11. The activity of V-ATPase is tightly controlled by its assembly of cytosolic V1 and membrane-bound V0 domains, as well as its trafficking between organelles. Previous studies have characterized the assembly mechanisms of V-ATPase. For example, the cryoelectron microscopy structure analysis of human V-ATPase showed that ATP6AP1 interacts with multiple subunits of V-ATPase, positioning it as a central hub for facilitating V-ATPase assembly12. The amino acid sensor mechanistic target of rapamycin complex 1 (mTORC1) was also identified to dynamically modulate the assembly/disassembly of V-ATPase in response to nutrient variations13. However, the mechanisms underlying V-ATPase trafficking to the specific organelle such as lysosomes remain largely unclear. Elucidating the specific host factors governing V-ATPase trafficking and deciphering the underlying mechanisms would significantly enhance our understanding on V-ATPase-mediated lysosomal acidification and its regulatory dynamics. Such insights could provide potential intervention targets and strategies against infectious diseases through improving host V-ATPase-dependent intrinsic immunity.

The Rab family of small GTPases serves as a highly conserved molecular switch governing vesicle budding, trafficking, and fusion in eukaryotic membrane trafficking systems, and thus has emerged as a pivotal regulatory hub that orchestrates multiple stages of host anti-infection immunity14, 15. However, the role of Rab proteins in regulating V-ATPase trafficking and lysosomal acidification, as well as the underlying mechanisms remain largely unexplored. Here, we demonstrate that Rab14 is essential for delivering V-ATPase to lysosomes for promoting acidification, thus functioning as a broad-spectrum antimicrobial factor against bacterial and viral pathogens. Mechanistic studies demonstrated that Rab14 competitively binds to calcium/calmodulin-dependent protein kinase type 2 delta (CAMK2D) with V0a1, the critical subunit protein determining V-ATPase localization16, followed by inhibiting CAMK2D-catalyzed V0a1 phosphorylation, ultimately eliciting V0a1 interaction with the COPⅡ complex to mediate the trafficking of V-ATPase from the endoplasmic reticulum (ER) to the Golgi apparatus and lysosomes. These data reveal that Rab14 is a broad-spectrum host restriction factor promoting V-ATPase trafficking and lysosomal acidification, suggesting that the Rab14-CAMK2D-V-ATPase axis serves as a promising target for treating infectious diseases.

Results
Rab14 is required for lysosomal acidification and pathogen clearance
To identify the critical Rab protein modulating lysosomal acidification during infections, we screened Rab proteins highly expressed in macrophages (> 50 nTPM, Supplementary Data 1), given their essential role as the primary immune defense against intracellular pathogens17. Then, the intracellular bacterium Mycobacterium smegmatis dually labeled with the pH-sensitive pHrodo fluorescent dye and the pH-insensitive Alexa Fluor 488 was used to infect macrophages with Rab gene knockdown for lysosomal pH analysis (Supplementary Fig. 1a, b and Supplementary Fig. 2). Interestingly, our system recapitulated the role of Rab32, which was reported to promote lysosomal acidification in osteoclasts18, confirming the system’s validity. Moreover, we also observed a significant increase in lysosomal pH upon Rab14 knockdown (Fig. 1a), indicating that Rab14 is a previously unrecognized critical factor in promoting lysosomal acidification. Additionally, Rab2, a protein that has been reported to have overlapping functions with Rab14 in promoting autophagosome-lysosome fusion in Madin-Darby canine kidney (MDCK) epithelial cells19, did not exhibit an effect on lysosomal pH in our experimental system. Thus, we focused on Rab14 to further explore its regulatory roles in lysosomal acidification. To further investigate whether the promoting effect of Rab14 on lysosomal acidification is unique to M. smegmatis infection, we constructed Rab14 knockout (Rab14-/-) macrophages (Supplementary Fig. 3a), and then infected wild-type (Rab14+/+) and Rab14-/- macrophages with another intracellular bacterium Listeria monocytogenes, as well as two types of viruses including DNA virus HSV-1 and RNA virus SeV. Through examining the fluorescence intensity of LAMP1 and LysoTracker, which are two biomarkers indicating total lysosomes and acidified lysosomes, respectively20, 21, we found that while the LysoTracker fluorescence intensity was much higher in infected Rab14+/+ macrophages than that in infected Rab14-/- macrophages, the LAMP1 intensity remained comparable between the two group (Fig. 1b–d), suggesting that Rab14 depletion specifically compromises lysosomal acidification without affecting lysosomal number. Moreover, we performed live-cell imaging assays using the LysoSensor Yellow/Blue probe, which mainly produces yellow fluorescence in acidic lysosomes (low pH), but blue fluorescence in less acidic lysosomes (high pH)22. The results demonstrated that compared with Rab14-/- macrophages, Rab14+/+ macrophages exhibited stronger yellow fluorescence but weaker blue fluorescence (Supplementary Fig. 3b), indicating a lower lysosomal pH in Rab14+/+ macrophages than that in Rab14-/- macrophages. Taken together, Rab14 is required for lysosomal acidification during a broad range of pathogen infections.

Lysosomal acidification is essential for the maturation and activation of lysosomal hydrolytic enzymes, especially the broadly specific aspartyl protease cathepsin D (CTSD)23. Consistent with the defective lysosomal acidification in Rab14-deficient macrophages, we observed decreased mature CTSD levels in Rab14-/- macrophages infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV than that in infected Rab14+/+ macrophages (Fig. 1e–h). In vitro CTSD activity assays also confirmed a marked reduction in proteolytic activity in Rab14-/- macrophages compared to Rab14+/+ macrophages, both under uninfected conditions and following infection with bacterial or viral pathogens (Supplementary Fig. 3c). Furthermore, as lysosomal acidification is critical for pathogen clearance, we detected the intracellular survival of pathogens in Rab14+/+ and Rab14-/- macrophages. The results showed that Rab14-/- macrophages exhibited increased intracellular pathogen survival than Rab14+/+ macrophages (Fig. 1i–l), indicating that Rab14 contributes to pathogen clearance. Moreover, it should be mentioned that lysosomal acidification is involved in multiple cellular processes including autophagy24, and we expectedly found an increased protein level of the 16-kD lipidated autophagosome-bound form of LC3B (LC3-II) in Rab14-/- macrophages infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV than that in infected Rab14+/+ macrophages, while the observed difference was abrogated by the autophagy flux inhibitor bafilomycin A1 (Baf)25, suggesting a disrupted autophagy flux in Rab14-/- macrophages (Supplementary Fig. 4a–d). Consistently, pathogen intracellular survival analysis demonstrated that Rab14 promotes the elimination of pathogens in a manner dependent on autophagy (Supplementary Fig. 4e–h). Collectively, Rab14 serves as an essential host restriction factor that orchestrates lysosomal acidification to promote pathogen clearance.

Rab14 elicits the lysosomal targeting of V-ATPase to promote lysosomal acidification
Next, we sought to investigate the mechanism by which Rab14 promotes lysosomal acidification. Given that V-ATPase is the master regulator of lysosomal acidification10, we wondered whether Rab14 affects the activation of V-ATPase to promote the acidification of lysosomes. V-ATPase is a multisubunit protein complex consisting of the cytosolic V1 domain and the lysosomal membrane-embedded V0 domain. The V1 domain is composed of eight different subunits (A, B, C, D, E, F, G, H) and is in charge of ATP hydrolysis, whereas the V0 domain contains six different subunits (a, d, e, c, c’, c’’) and is responsible for the translocation of protons from the cytoplasm to the organelle lumen16. The reversible assembly of V1/V0 on the lysosomal membrane is essential for regulating lysosomal pH. Prior to this, the V0 sector needs to be transported to the lysosomal membrane, which is primarily regulated by the subunit a of V0 (V0a)16. As Rab14 is a small GTPase contributing to membrane trafficking26-28, we speculated that Rab14 might be involved in the trafficking of V0a to lysosomes. There are four isoforms of V0a (a1-a4). Among them, V0a1, V0a2, and V0a3, but not V0a4, can be expressed in macrophages (https://www.proteinatlas.org/). Therefore, we detected the subcellular localization of V0a1, V0a2, and V0a3 in Rab14+/+ and Rab14-/- cells, respectively. The double-immunofluorescence labeling analysis revealed strong colocalization of V0a1 with LAMP1-positive lysosomes in Rab14+/+ cells, whereas in Rab14-/- cells, V0a1 primarily colocalized with the ER-integral protein calnexin (Fig. 2a–e). Moreover, V0a1 showed minimal colocalization with GM130 (the biomarker for Golgi apparatus), Rab5 (the biomarker for early endosomes), and Rab7 (the biomarker for late endosomes) in Rab14+/+ cells, and these colocalizations further reduced in Rab14-/- cells (Fig. 2a–e). Furthermore, we isolated ER and lysosomal fractions from Rab14+/+ and Rab14-/- cells to examine the distribution of V0a1, and consistently found that V0a1 was predominantly enriched on lysosomes in Rab14+/+ cells, whereas it was largely retained in the ER in Rab14-/- cells (Supplementary Fig. 5a). Unlike the different pattern of V0a1 subcellular localization in Rab14+/+ and Rab14-/- cells, there was no significant difference in the subcellular localization of V0a2 and V0a3 between Rab14+/+ and Rab14-/- cells (Supplementary Fig. 5b–k). Taken together, Rab14 promotes the lysosomal targeting of V0a1.

To verify the regulatory role of Rab14 on the lysosomal localization of V0a1 during pathogen infections, we detected the colocalization of V0a1 and lysosomes in Rab14+/+ and Rab14-/- cells infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV. The results showed that the above pathogen infections could increase the trafficking of V0a1 to lysosomes in Rab14+/+ cells, while this effect is largely reduced in Rab14-/- cells (Fig. 2f; Supplementary Movies 1, 2). Analysis of V0a1 levels in purified lysosomes also demonstrated a significantly higher enrichment of V0a1 on lysosomes from Rab14+/+ cells than those from Rab14-/- cells (Supplementary Fig. 6a). To further confirm whether Rab14 promotes lysosomal acidification in a manner dependent on V0a1, we employed siRNA to knock down V0a1 genes in Rab14+/+ and Rab14-/- cells (Supplementary Fig. 6b) and then examined the fluorescence intensity of LysoTracker. We found that V0a1 knockdown eliminated the effects of Rab14 promoting lysosomal acidification during pathogen infections (Supplementary Fig. 6c). Finally, pathogen intracellular survival analysis also confirmed that Rab14 promotes pathogen clearance depending on V0a1 (Supplementary Fig. 6d–g). Altogether, these results indicate that Rab14 facilitates V-ATPase lysosomal targeting to promote lysosomal acidification and pathogen clearance.

Rab14 suppresses CAMK2D-mediated V0a1 phosphorylation to promote V0a1 trafficking to lysosomes
Rab GTPases usually regulate protein trafficking by direct interaction29. Thus, we firstly examined whether Rab14 can interact with V0a1 to promote its transport to lysosomes. The co-immunoprecipitation analysis showed that there is no interaction between Rab14 and V0a1 (Supplementary Fig. 7a), indicating that Rab14 modulates V0a1 trafficking in a way independent of their interaction. Studies have reported that the protein post-translational modification (PTM) of V0a1 is important for its trafficking and localization. For example, the glycosylation of V0a1 is essential for its maturation and the ensuing ER-to-lysosome delivery30, while our data showed no molecular weight differences in V0a1 between Rab14+/+ and Rab14-/- cells that would indicate altered glycosylation (Fig. 3a). Furthermore, V0a1 requires palmitoylation for its trafficking from the sorting endosome to the late endosomal/lysosomal membrane31, while Rab14 mainly modulates the delivery of V0a1 from ER to Golgi (Fig. 2a), which is a different trafficking way independent of the endosomal sorting pathway. Consistently, the palmitoylation assay demonstrated no significant difference in V0a1 palmitoylation levels between Rab14+/+ and Rab14-/- cells (Supplementary Fig. 7b). Taken together, these results suggest that there might be unrecognized PTMs of V0a1 modulated by Rab14 to govern V0a1 trafficking.

Given that Rab14 lacks enzymatic activity for PTMs, we speculated that Rab14 might recruit other proteins to modulate V0a1 PTM. By conducting immunoprecipitation-mass spectrometry, we obtained 43 proteins potentially interacted with Rab14 (Supplementary Fig. 7c; Supplementary Data 2). Among them, TRIM21 is an E3 ubiquitin ligase mediating protein ubiquitination, and CAMK2D is a serine/threonine kinase catalyzing protein phosphorylation32, 33. Then, we detected whether Rab14 modulates the ubiquitination and phosphorylation of V0a1. In vivo ubiquitination assays showed that V0a1 was ubiquitinated at comparable levels in Rab14+/+ and Rab14-/- cells (Supplementary Fig. 7d), indicating that Rab14 does not modulate the ubiquitination of V0a1. However, we interestingly observed a higher serine, but not threonine, phosphorylation level of V0a1 in Rab14-/- cells than that in Rab14+/+ cells (Fig. 3a), indicating that Rab14 suppresses the phosphorylation of V0a1. To further investigate whether CAMK2D is involved in the Rab14-mediated suppression of V0a1 phosphorylation, we firstly detected the interactions among Rab14, CAMK2D, and V0a1. We found that Rab14 could interact with CAMK2D (Supplementary Fig. 7e). Interestingly, CAMK2D specifically bound to V0a1, but not V0a2 or V0a3 (Supplementary Fig. 8a). We then conducted molecular docking analysis and revealed that CAMK2D bound to the Arg 179 and Arg 198 sites of V0a1, two residues that are not conserved in V0a2 or V0a3 (Supplementary Fig. 8b, c). Moreover, the V0a1-CAMK2D affinity was stronger in Rab14-/- cells than that in Rab14+/+ cells, indicating a competition between Rab14 and V0a1 for CAMK2D (Supplementary Fig. 8d). Through further conducting the in vitro competition binding assay, we verified that Rab14 competitively inhibits the interaction between V0a1 and CAMK2D (Fig. 3b). We then explored the molecular mechanism by which Rab14 and V0a1 competitively bind to CAMK2D. Through constructing the truncated variants of CAMK2D, we found that both Rab14 and V0a1 interacted with the kinase domain of CAMK2D (Supplementary Fig. 8e, f), which is the critical region mediating substrate phosphorylation34. Furthermore, the in vitro phosphorylation assay showed that V0a1, but not V0a2 or V0a3, was phosphorylated by CAMK2D (Supplementary Fig. 8g), while this phenomenon was largely abolished by Rab14 (Fig. 3c). Moreover, CAMK2D deficiency resulted in the loss of V0a1 phosphorylation in Rab14-/- cells (Fig. 3d). Collectively, these results suggest that Rab14 competitively interacts with CAMK2D to suppress CAMK2D-mediated V0a1 phosphorylation. Next, we explored the role of CAMK2D-mediated V0a1 phosphorylation in Rab14-governed V0a1 trafficking. We found that the Rab14 knockout-caused high colocalization of V0a1 with ER and low colocalization of V0a1 with lysosomes could be reversed by CAMK2D deletion (Fig. 3e, f), indicating that CAMK2D functions as a negative regulator in Rab14-promoted V0a1 trafficking to lysosomes. Altogether, Rab14 promotes V-ATPase lysosomal delivery by antagonizing CAMK2D-mediated V0a1 phosphorylation.

Rab14-mediated inhibition of V0a1 phosphorylation enhances its binding to the COPⅡ complex
Next, we investigated the detailed mechanism by which enhanced V0a1 phosphorylation impairs its transport from ER to Golgi and then to lysosomes in Rab14-/- cells. Through examining the subcellular localization of CAMK2D by confocal microscopy analysis, we found that CAMK2D exhibits a diffuse cytoplasmic distribution, and this localization pattern remains unchanged in Rab14-/- cells (Supplementary Fig. 9a), indicating that Rab14 modulates V0a1 trafficking independent of CAMK2D localization. Studies reported that the coat protein complex Ⅱ (COPⅡ), which is composed of secretion-associated Ras-related GTPase 1 (Sar1), Sec23/Sec24, and Sec13/Sec31 subunits, plays a crucial role in the delivery of V0 complex from ER to Golgi35. Since S-palmitoylation is essential for COPII-mediated anterograde ER-to-Golgi trafficking36, we thus examined whether Rab14 deficiency alters this modification. Our results showed that the S-palmitoylation levels of COPII-associated proteins were comparable between Rab14+/+ and Rab14-/- cells (Supplementary Fig. 9b), indicating that Rab14 is not involved in regulating the palmitoylation of the COPII complex. Moreover, within the COPII coat, Sec24 is primarily responsible for cargo selection through directly recognizing and binding cargo proteins37. Among the four isoforms of Sec24, including Sec24A, Sec24B, Sec24C, and Sec24D, Sec24C was indicated to be involved in the ER-to-Golgi transport of V0 subunits38. Based on the above evidence, we hypothesized that whether V0a1 phosphorylation influences its interaction with Sec24C. Through conducting the coimmunoprecipitation assay of V0a1 and Sec24C, we demonstrated a specific interaction of these two proteins in Rab14+/+ cells, but not in Rab14-/- cells. Intriguingly, the deletion of CAMK2D, which is responsible for mediating the phosphorylation of V0a1, restored the interaction between V0a1 and Sec24C (Fig. 4a), indicating an inhibitory effect of V0a1 phosphorylation on its binding to Sec24C. To further confirm this result, we analyzed the specific phosphorylation site of V0a1 to determine whether its mutation could reverse the V0a1-Sec24C interaction. Through using the GPS 5.0 database (https://gps.biocuckoo.cn/)39, we identified a total of four potential phosphorylation sites on V0a1, including serine 19 (S19), threonine 182 (T182), threonine 371 (T371), and serine 432 (S432) (Supplementary Fig. 9c). We subsequently mutated these residues to alanine (A), and found that while the threonine phosphorylation level of V0a1 was unaffected by CAMK2D, the S19A mutation abolished CAMK2D-mediated serine phosphorylation of V0a1 (Supplementary Fig. 9d). Consistently, the phosphorylation of V0a1 in Rab14-/- cells was also abrogated by the S19A mutation (Supplementary Fig. 9e). Thus, Rab14 inhibits CAMK2D-mediated V0a1 phosphorylation at the S19 site. To this end, we generated a phosphospecific antibody against the V0a1 S19 site. Through in vitro and in vivo phosphorylation assays using this antibody, we confirmed that CAMK2D phosphorylates V0a1 at S19, while Rab14 inhibits this process (Supplementary Fig. 9f, g). Then, we examined the binding of V0a1 or its S19A mutant to Sec24C in Rab14+/+ and Rab14-/- cells, respectively. The result showed that the S19A mutation restored the interaction between V0a1 and Sec24C in Rab14-/- cells (Fig. 4b). Likewise, the colocalization analysis also demonstrated that the V0a1S19A mutant exhibited a higher colocalization efficiency with Sec24C than the wild-type V0a1 in Rab14-/- cells (Fig. 4c). Accordingly, mutating the S19 residue in V0a1 increased the localization of V0a1 in lysosomes, while decreasing its retention in ER in Rab14-/- cells (Fig. 4d, e). Collectively, Rab14 inhibits CAMK2D-mediated V0a1 phosphorylation to enhance V0a1 binding to COPⅡ complex, thereby eliciting V0a1 trafficking to lysosomes.

Pathogen infection upregulates GTP-bound Rab14 to inhibit CAMK2D-mediated V0a1 phosphorylation
Next, we explored the regulatory dynamics of the Rab14-CAMK2D-V0a1 axis during pathogen infections. As reported, Rab proteins cycle between their active GTP-bound and inactive GDP-bound states, with the GTP-bound conformation typically being crucial for pathogen infections40, 41. Through immunoprecipitating the active GTP-bound Rab14 using RUFY1, which specifically recognizes the constitutively active GTP-locked mutant of Rab1442, we observed increased GTP-bound Rab14 levels following infection with M. smegmatis, L. monocytogenes, HSV-1, or SeV (Fig. 5a–d). Given that Rab proteins in their GTP-bound state recruit various effector proteins to facilitate membrane trafficking, we hypothesized that increased GTP-bound Rab14 could enhance the interaction between Rab14 and CAMK2D during pathogen infections. Firstly, we detected the affinity between CAMK2D and Rab14 active or inactive mutant, and the result showed that CAMK2D preferred to bind to the GTP-bound Rab14 (Supplementary Fig. 10a). Then, we consistently observed increased Rab14-CAMK2D interactions during pathogen infections (Fig. 5e–h). Based on the findings that Rab14 competitively binds to CAMK2D to suppress CAMK2D-mediated V0a1 phosphorylation and thus promote V0a1 interacting with Sec24C (Figs. 3, 4), we proceeded to investigate the phosphorylation status of V0a1 and its interaction with Sec24C during infection with different pathogens in Rab14+/+, Rab14-/-, Rab14+/+Camk2d-/-, and Rab14-/-Camk2d-/- cells. Strikingly, V0a1 was significantly phosphorylated in Rab14-/- cells as compared to that in Rab14+/+ cells, and the low baseline phosphorylation of V0a1 in Rab14+/+ cells progressively declined over the course of infection (Supplementary Fig. 10b–e). Moreover, CAMK2D knockout resulted in a complete loss of V0a1 phosphorylation in both Rab14+/+ and Rab14-/- cells (Supplementary Fig. 10b–e). Conversely, Rab14-/- cells showed loss of V0a1-Sec24C interaction, whereas their binding increased progressively during infection in Rab14+/+ cells and remained strong in Rab14+/+Camk2d-/- and Rab14-/-Camk2d-/- cells (Supplementary Fig. 10b–e). Thus, these results indicate that Rab14 suppresses CAMK2D-mediated V0a1 phosphorylation to facilitate V0a1-Sec24C complex formation during pathogen infections. Finally, quantitative imaging analysis for V0a1-lysosome colocalization revealed that Rab14 promotes the trafficking of V0a1 to lysosomes in a manner dependent on CAMK2D (Fig. 5i). It should be pointed out that Rab14-promoted lysosomal localization of V0a1 was observed not only under infection conditions, but also in non-infection conditions, indicating that Rab14 also regulates the baseline level of lysosomal acidification. Altogether, our data suggest that upon pathogen infections, the active GTP-bound Rab14 increases to interact with CAMK2D, thus suppressing CAMK2D-mediated V0a1 phosphorylation to enhance V0a1 interaction with the COPⅡ complex, ultimately promoting V0a1 trafficking to lysosomes.

Rab14 promotes lysosomal acidification and pathogen clearance through CAMK2D
Given that Rab14 interacts with CAMK2D to facilitate the lysosomal targeting of V0a1, we then further investigated the role of CAMK2D in Rab14-promoted lysosomal acidification. Through examining the fluorescence intensity of LysoTracker in Rab14+/+, Rab14-/-, Rab14+/+Camk2d-/-, and Rab14-/-Camk2d-/- cells infected with M. smegmatis, L. monocytogenes, HSV-1, or SeV, we found that upon pathogen infections, Rab14-/- cells exhibited a lower LysoTracker fluorescence intensity compared to Rab14+/+ cells, while CAMK2D knockout enhanced the LysoTracker fluorescence in Rab14-/- cells, eliminating the phenotypic disparity between Rab14+/+ and Rab14-/- cells (Fig. 6a). Consistently, the lysosomal protease CTSD maturation analysis and CTSD activity assay revealed that Rab14 promotes the maturation and activation of CTSD in a manner dependent on CAMK2D (Fig. 6b–e, Supplementary Fig. 11a). Furthermore, we monitored the dynamic autophagy flux in the above four types of cell lines by using the mCherry-GFP-LC3 tandem reporter system, which manifests as red puncta within autolysosomes and yellow puncta in immature autophagosomes43. The results showed that although the number of the total LC3 puncta per cell was comparable in all cell lines upon pathogen infections, the ratio of yellow:total LC3 puncta was significantly increased in Rab14-/- cells than that in Rab14+/+, Rab14+/+Camk2d-/-, and Rab14-/-Camk2d-/- cells, indicating that Rab14 depends on CAMK2D to enhance autophagy flux during pathogen infections (Supplementary Fig. 12). A recent study reported that Rab14 promotes autophagy flux by facilitating homotypic fusion and protein sorting (HOPS) complex-mediated autophagosome-lysosome fusion in MDCK epithelial cells19. To determine whether the Rab14–CAMK2D axis regulates this process in macrophages, we examined the colocalization of LC3 with LAMP1 in Rab14+/+, Rab14-/-, Rab14+/+Camk2d-/-, and Rab14-/-Camk2d-/- macrophages under uninfected or infected conditions, and our results showed that the Rab14-CAMK2D axis did not affect the fusion between autophagosomes and lysosomes (Supplementary Fig. 13), suggesting that Rab14-CAMK2D axis promotes autophagy flux through facilitating lysosomal acidification without affecting the autophagosome-lysosome fusion. Finally, the pathogen intracellular survival assay demonstrated that Rab14 promotes the clearance of pathogens depending on CAMK2D (Fig. 6f). Collectively, CAMK2D is critical for Rab14-promoted lysosomal acidification and pathogen clearance.

To further verify whether CAMK2D participates in the modulation of Rab14-mediated lysosomal acidification and pathogen clearance depending on its kinase activity, we detected the LysoTracker fluorescence intensity and pathogen survival in Rab14+/+ and Rab14-/- cells with or without the treatment of KN-93 Phosphate (KN-93-P), which is a robust and specific inhibitor towards to CAMKⅡ family44. As expected, inhibiting the kinase activity of CAMK2D using KN-93-P increased the fluorescence intensity of LysoTracker and improved the clearance of pathogens in Rab14-/- cells, which levels were comparable to those in Rab14+/+ cells (Supplementary Fig. 14). Altogether, these results indicate that Rab14 targets CAMK2D kinase activity to promote lysosomal acidification and pathogen clearance.

Rab14-mediated inhibition of CAMK2D-catalyzed V0a1 phosphorylation is required for pathogen clearance in vivo
To further determine the physiological relevance of Rab14-mediated inhibition of CAMK2D-catalyzed V0a1 phosphorylation in pathogen clearance in vivo, we challenged Rab14flox/flox (Rab14fl/fl) and rab14fl/fl-Lyz2-Cre (rab14lyz2) mice with M. smegmatis, L. monocytogenes, HSV-1, or SeV in the absence or presence of CMK2D kinase activity inhibitor KN-93-P. Through analyzing the pathogen burdens in different organs during infections, we found that rab14lyz2 mice displayed higher bacterial loads or virus titers than Rab14fl/fl mice, while KN-93-P treatment markedly reduced pathogen burdens in rab14lyz2 mice, restoring them to levels comparable to those observed in Rab14fl/fl mice (Fig. 7a–d). Consistently, histopathological analysis demonstrated that Rab14 deficiency led to increased inflammatory cell infiltration in the lungs of mice infected with M. smegmatis, HSV-1, or SeV, as well as in the livers of mice infected with L. monocytogenes, but the administration of KN-93-P attenuated these inflammatory responses (Fig. 7e–h). Thus, these results indicate that Rab14 functions as a critical host restriction factor that broadly defends against bacterial and viral infections through suppressing CAMK2D kinase activity towards V0a1.

In summary, our study demonstrates that upon pathogen infections, the active GTP-bound Rab14 increases and binds to CAMK2D, disrupting the CAMK2D-V0a1 interaction and CAMK2D-mediated phosphorylation of V0a1 at Ser19, thereby enabling V0a1 to engage with Sec24C in the COPII complex to facilitate the anterograde transport of the V0 complex from the ER to the Golgi apparatus and then to lysosomes. Ultimately, the V0 complex assembles with the V1 complex to form a functional V-ATPase for mediating lysosomal acidification and pathogen clearance (Supplementary Fig. 15).

Methods
Research ethics board
All animal breeding and procedures were performed in accordance with the instructional guidelines of the China Council on Animal Care, and were approved by the Biomedical Research Ethics Committee of the Institute of Microbiology, Chinese Academy of Sciences, and the Beijing Chest Hospital, Capital Medical University (HP-SQIMCAS2025159).

Mouse models
Rab14fl/fl mice and rab14fl/fl-Lyz2-Cre (rab14lyz2) mice with Rab14 deficiency in myeloid cell lineage (monocytes, mature macrophages, and granulocytes) were generated as described previously49. Briefly, Rab14fl/fl mice and Lyz2-Cre mice were obtained from Biocytogen (Beijing, China) and Jackson Laboratory, respectively. Rab14fl/fl mice were crossed with Lyz2-Cre mice to generate F1 rab14fl/+-Lyz2-Cre mice, followed by crossing with Rab14fl/fl mice to generate rab14lyz2 mice. All mice were housed and bred in a specific pathogen-free (SPF) facility using standard humane animal husbandry protocols (12 h light/dark cycle, 50% relative humidity, at 25–27°C, free access to food and tap water), which were approved by the animal care and use committee of the Institute of Microbiology (Chinese Academy of Sciences).

Rab14fl/fl mice and rab14lyz2 mice aged 6–8 weeks and sex-matched are infected with the indicated pathogens. For M. smegmatis infection, mice were intratracheally infected with 1 × 108 CFU of M. smegmatis mc2155 strain per mouse, concurrently with 5 mg/kg KN-93-P treatment via intraperitoneal injection once every 3 days. After 7 days, lungs and spleens were homogenized with a FastPrep-24 System (MP Biomedicals) for CFU counting, and lungs were fixed in 10% formalin and embedded in paraffin for hematoxylin and eosin staining. The tissue slides were scanned with Aperio CS2 (Leica Biosystems), and the histopathology of tissues was analyzed by ImageScope (v12.3.3.7014) software. For L. monocytogenes infection, mice were intraperitoneally infected with 2.5 × 105 CFU of L. monocytogenes per mouse and treated with KN-93-P as above. After 3 days of infection, livers and spleens were homogenized for assessing bacterial numbers in organs, and livers were subjected to section for pathological analysis. For HSV-1 and SeV infection, mice were intravenously infected with 2 × 107 PFU of each virus per mouse and treated with KN-93-P as above. After 2 days of infection, lungs and spleens were homogenized for detecting virus titers, and lungs were subjected to section for pathological analysis.

Bacterial strains, viruses, and plasmids
Mycobacterium smegmatis mc2155 (ATCC, 7010084) was grown in Middlebrook 7H9 medium (BD, 271310) supplemented with 10% oleic acid-albumin-dexrose-catalase (OADC) and 0.05% Tween-80 (G-CLONE, CS9029), or on Middlebrook 7H10 agar (BD, 262710) supplemented with 10% OADC. Listeria monocytogenes (ATCC, 19115) was cultured in liquid brain heart infusion (BHI; ELITE-MEDIA, m330-02) or BHI agar plates overnight at 37°C. HSV-1 was a kind gift of Xin Ye (Institute of Microbiology, Chinese Academy of Sciences, Beijing) and was propagated in Vero cells (ATCC, CCL-81). SeV was a kind gift of George Fu Gao (Institute of Microbiology, Chinese Academy of Sciences, Beijing) and was propagated in 10-day-old SPF chicken embryos.

For protein expression in mammalian cells, Rab14 was cloned into pcDNA6A (with MYC-tag) (Invitrogen, V22120), pEGFP-C1 (with GFP-tag) (Clontech, V012024), or p3xFlag-CMV14 (with Flag-tag) vector (Sigma-Aldrich, E7908); CAMK2D and its truncated mutants were cloned into p3xFlag-CMV14 (with Flag-tag) or pEGFP-C1 (with GFP-tag) vector; V0a1 and its mutants, V0a2, and V0a3 were cloned into pEGFP-C1 (with GFP-tag) vector. For protein expression in bacteria, Rab14 or RUFY1 was cloned into pGEX-6p-1 (with GST-tag) vector (GE Healthcare, 28-9546-48), CAMK2D was cloned into pET30a (with His-tag) vector (Novagen, 69909). All the plasmids were sequenced at the Beijing Genomics Institute (BGI) for verification. All of the plasmids and primers used in this study were detailed in Supplementary Data 3.

Antibodies and reagents
All antibodies were used according to the manufacturers’ instructions. The commercial antibodies used in this study were as follows: anti-Rab14 (Santa Cruz Biotechnology, sc-271401, 1:100 for immunoprecipitation, 1:1,000 for immunoblotting), anti-CTSD (Abclonal, A13292, 1:2,000 for immunoblotting), anti-cleaved CTSD LC (Immunoway, YC0038, 1:2,000 for immunoblotting), anti-LC3B (Sigma-Aldrich, L7543, 1:3,000 for immunoblotting), anti-calnexin (Santa Cruz Biotechnology, sc-23954, 1:200 for immunofluorescence), anti-GM130 (Abcam, ab52649, 1:200 for immunofluorescence), anti-Rab5 (Abcam, ab218624, 1:100 for immunofluorescence), anti-Rab7 (Abcam, ab137029, 1:100 for immunofluorescence), anti-LAMP1 (Cell Signaling Technology, 99437, 1:100 for immunofluorescence), anti-p-Ser (Abcam, ab9332, 1:1,000 for immunoblotting), anti-p-Thr (Cell Signaling Technology, 9386, 1:1,000 for immunoblotting), anti-V0a1 (Proteintech, 13828-1-AP, 1:100 for immunoprecipitation, 1:1,000 for immunoblotting), anti-CAMK2D (Abclonal, A9196, 1:2,000 for immunoblotting), anti-Sar1 (Immunoway, YN0749, 1:2,000 for immunoblotting), anti-Sec23A (Immunoway, YN3350, 1:2,000 for immunoblotting), anti-Sec24A (Immunoway, YN6048, 1:2,000 for immunoblotting), anti-Sec24C (Abclonal, A10797, 1:2,000 for immunoblotting), anti-Sec31A (Immunoway, YN5979, 1:2,000 for immunoblotting), anti-TUBA1A (Sigma-Aldrich, T6199, 1:5,000 for immunoblotting), anti-GAPDH (Santa Cruz Biotechnology, sc-25778, 1:5,000 for immunoblotting), anti-Myc (MBL International, M047-7, 1:4,000 for immunoblotting), anti-Myc (Cell Signaling Technologies, 2276S, 1:200 for immunofluorescence), anti-Flag (MBL International, PM020-7, 1:4,000 for immunoblotting), anti-GFP (MBL International, 598-7, 1:4,000 for immunoblotting), anti-GST (MBL International, PM013-7, 1:4,000 for immunoblotting), anti-His (Abclonal, AE003, 1:4,000 for immunoblotting), HRP-labeled goat anti-rabbit IgG (ZSGB Biotech, ZB-2306, 1:10,000 for immunoblotting), HRP-labeled goat anti-mouse IgG (ZSGB Biotech, ZB-2305, 1:10,000 for immunoblotting), Alexa Fluor 594-labeled goat anti-rabbit IgG (ZSGB Biotech, ZF0516, 1:200 for immunofluorescence), Alexa Fluor 594-labeled goat anti-mouse IgG (ZSGB Biotech, ZF0513, 1:200 for immunofluorescence), and Alexa Fluor 488-labeled goat anti-rabbit IgG (CHEJETER, ZF-0511, 1:200 for immunofluorescence). The phosphospecific antibody against p-V0a1 (Ser19) was produced as described previously57, 58. Briefly, a 16-amino acid peptide encompassing phospho-Ser19 was synthesized and conjugated to keyhole limpet hemocyanin (KLH) for immunization in rabbits. Thereafter, the antibody was affinity-purified from the serum using the same phospho-peptide. The purified antibody was validated by immunoblotting at a working concentration of 5 µg/mL, demonstrating a specific signal against GFP-V0a1, but not the GFP-V0a1S19A mutant.

The commercial reagents used in this study were as follows: Bafilomycin A1 (Baf; Selleck, S1413), KN-93 phosphate (Selleck, S7423), LysoTracker (Invitrogen, L7528), and LysoSensor Yellow/Blue probe (Invitrogen, L7545).

Cell culture and transfection
HEK293T cells (ATCC, CRL-3216), HeLa cells (ATCC, CCL-2), Vero cells (ATCC, CCL-81), and RAW264.7 cells (ATCC, TIB-71) were obtained from the American type culture collection (ATCC). The above cell lines were all cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, C11995500BT) with 10% fetal bovine serum (FBS; Gibco, 16000-044) at 37ºC in a humidified 5% CO2 incubator. In related experiments, the transfection of HEK293T or HeLa cells was carried out with KunGre™ Liposomal Transfection Reagent (Greact, GT201-03) according to the manufacturers’ instructions.

Recombinant protein expression and purification
For prokaryotic expression of GST, GST-Rab14, GST-RUFY1, and His-CAMK2D, E. coli BL21 (DE3) strains harboring GST or His derivatives were grown in LB medium supplemented with the indicated antibiotics at 37°C until the OD600 = 0.6. Then, 0.1 mM Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the cultures to induce the expression of proteins at 16°C for 16 hours. Bacteria were then harvested by centrifugation at 6,500 ×g for 10 minutes and suspended in a buffer containing 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. Subsequently, cells were broken using Low-temperature Ultra-high Pressure Continuous Flow Cell Disrupters (JNBIO) for collecting the supernatants. Glutathione Sepharose 4B (GE Healthcare, 17-0756-05) and Ni-NTA Agarose (Qiagen, 30230) were added to the supernatants for purification of GST- or His-tagged proteins, respectively. For eukaryotic expression and purification of GFP-tagged V0a1 or its mutants, Hela cells were transfected with the indicated plasmids for 24 hours and were then lysed in the RIPA lysis buffer (Beyotime Biotechnology, P0013D) supplemented with 1% protease inhibitor cocktail (Bimake, B14001) at 4°C for 10 minutes. The cell lysates were then incubated with anti-GFP-Nanoab-Agarose (LABLEAD, GNA-50-1000) at 4°C for 4 hours. After extensively washing with the lysis buffer, the GFP-tagged proteins were eluted by 50 μL elution buffer (0.2 mM glycine, adjust pH to 2.5 by HCl) and neutralized by 5 μL neutralization buffer (1.0 M Tris-HCl, pH 10.4). Protein concentrations were determined by the BCA protein assay (Beyotime, P0009) and protein purity was examined by SDS-PAGE analysis.

Establishment of knockout and knockdown cell lines
RAW264.7 cells and Hela cells with Rab14 or CAMK2D knocking out were generated using the CRISPR/Cas9 gene-editing system. Specifically, the sgRNA sequences targeting Rab14 or CAMK2D (see Supplementary Data 3) were predicted by the CRISPR-Gold (https://crisprgold.mdc-berlin.de/) and were then cloned into the pSpCas9(BB)-2A-GFP vector (Addgene, 48138). Next, RAW264.7 cells and Hela cells were transfected with the specific recombinant plasmid for 24 hours. The single cell with green fluorescence was sorted with BD FACSAria III cell sorter (BD Biosciences) and was then identified by immunoblotting analysis with the antibody against Rab14 or CAMK2D.

For knocking down Rab genes in RAW264.7 cells, the siRNA sequences targeting the specific Rab (see Supplementary Data 3) were designed by BLOCK-iT™ RNAi Designer. Then, 40 pM siRNAs targeting each Rab gene were transfected into RAW264.7 cells by using KunGreTM Liposomal Transfection Reagent (Greact, GT201-03) according to the manufacturer’s instructions. After 12 hours, cells were re-transfected with the sgRNAs and were then further incubated for another 12 hours. The knocking down efficiency of Rab genes was verified by the quantitative real-time polymerase chain reaction (qPCR) analysis. V0a1-knocking down Rab14+/+ and Rab14-/- RAW264.7 cells were generated in the same as above and were then identified by immunoblotting analysis with the antibody against V0a1.

qPCR analysis
RAW264.7 cells transfected with Rab siRNAs were collected for total RNA extraction using the Total RNA Extraction Kit (Dongsheng, R1061). Then, the RNA was reverse-transcribed into cDNA using the Hifair First Strand cDNA Synthesis SuperMix (YEASEN, 11141ES60). The cDNA was then analyzed by qPCR using Hieff qPCR SYBR Green Master Mix (YEASEN, 11202ES08) on an ABI 7500 system (Applied Biosystems). Quantitative expression of the targeted genes was normalized to Gapdh and analyzed by the 2−ΔΔCT method. All qPCR primers are listed in Supplementary Data 3.

Establishment of RAW264.7 cells stably expressing mCherry-GFP-LC3
RAW264.7 cells stably expressing mCherry-GFP-LC3 were generated as described previously49. Briefly, PMSCV puro-mCherry-GFP-LC3 and the two packaging plasmids including pVSV-G and pCMV-Gag-Pol were co-transfected to HEK293T cells for obtaining lentivirus. Then, the lentiviral supernatant was collected at 48 hours post-transfection, filtered with a 0.45-μm filter, and concentrated by Lentivirus Concentration Solution (YEASEN, 41101ES50). The medium containing lentivirus was used to infect RAW264.7 cells, and the cells stably expressing mCherry-GFP-LC3 were selected using 2 μg/mL puromycin (Selleck, s7417). The expression of mCherry-GFP-LC3 was verified by immunofluorescence microscopy.

Measurement of phagolysosomal pH
The pH of pathogen-containing phagolysosomes was detected as described previously59. Briefly, the model pathogen M. smegmatis was first labeled with 20 μM pHrodo Red succinimidyl ester (Invitrogen, P36600) at 37°C for 1 hour. Next, the labelled bacteria were washed three times with Middlebrook 7H9 media containing 0.05% Tween-80 and were then labeled with 25 μg/mL Alexa Fluor 488 succinimidyl ester (Invitrogen, A20000) at 37°C for 1 hour. After washing again, bacterial pellets were resuspended in DMEM containing 0.05% Tween-80. Thereafter, RAW264.7 cells were infected with the double-labeled M. smegmatis at a multiplicity of infection (MOI) of 10 for 2 hours and were then washed with 1 × PBS to remove any non-internalized bacteria. The infection was incubated for another 2 hours at 37°C with 5% CO2, and the fluorescence intensity in the RAW264.7 cells was analyzed by Flow cytometer (BD LSRFortessa SORP) and FlowJo (v10) software. The phagolysosomal pH was calculated by the mean fluorescence ratios of pHrodo and Alexa Fluor 488.

Pathogen infections of macrophages
RAW264.7 cells were seeded at 1 × 106 cells per well in 6-well plates and cultured at 37ºC with 5% CO2 overnight. For M. smegmatis infection, frozen bacteria that have grown to mid-logarithmic phase were thawed and were then centrifuged and resuspended in DMEM medium with 0.05% Tween-80 to infect RAW264.7 cells at a MOI of 10. After 2 hours of infection, cells were washed three times with 1 × PBS to exclude non-internalized bacteria and were then incubated with the fresh DMEM medium supplemented with 10 μg/mL gentamicin to kill non-washed extracellular bacteria. At the indicated time points, cells were harvested for colony-forming unit (CFU) counting analysis, immunoblotting analysis, and immunofluorescence analysis. For L. monocytogenes infection, bacteria were grown in BHI broth to mid-log phase and were then resuspended in DMEM medium to infect RAW264.7 cells at a MOI of 5. After 1 hour of infection, cells were washed with 1 × PBS to exclude non-internalized bacteria and were then incubated with the fresh DMEM medium supplemented with 10 μg/mL gentamicin until the indicated time points for further CFU, immunoblotting, and immunofluorescence analysis. For viral infection, the DNA virus HSV-1 and RNA virus SeV were both used to infect RAW264.7 cells at a MOI of 1. At the indicated time points, the cells were collected for virus copy number, immunoblotting, and immunofluorescence analysis. For inhibition of CAMK2D or autophagy flux, cells were treated with 10 μM KN-93-P or 50 nM Baf, respectively, and the control groups were treated with equal volume of dimethyl sulfoxide.

CTSD activity assay
CTSD activity was measured using a commercial assay kit (Abcam, ab65302). Briefly, harvested cells were lysed in chilled buffer, and the lysates were incubated with the substrate-containing reaction mix at 37°C for 1–2 hours, protected from light. Fluorescence was measured at Ex/Em = 328/460 nm using a microplate reader. Enzyme activity was determined based on the fold change in relative fluorescence units (RFU) of treated samples compared to control samples.

CFU counting analysis of bacteria and plaque assay of virus
For CFU counting analysis of M. smegmatis or L. monocytogenes, infected macrophages or tissues were lysed in 1 × PBS containing 0.05% SDS. Then, the cell lysates were plated by serial dilution on 7H10 agar plates for 72 hours or on BHI agar plates for 24 hours, respectively. For plaque assay of virus, the homogenates of lungs or spleens were harvested and diluted to infect confluent Vero cells. At 2 hours post infection, the supernatant was removed and the infected Vero cells were overlaied with a mixed medium of 2% low melting point agarose (YEASEN, 10214ES08) and 2% FBS at a ratio of 1:1. After 72 hours post infection, the overlay was removed and the cells were fixed with 4% paraformaldehyde (Sigma-Aldrich, 441244) for 15 minutes and stained with 0.1% crystal violetin (Huaaobio, 01-174) for 30 minutes. Then, the plaques were counted, averaged, and multiplied by the dilution factor to determine viral titers as PFU/mL. Virus copy numbers were determined by qPCR using the corresponding primers (Supplementary Data 3).

ER and lysosome extraction
ER and lysosomal fractions were isolated using the Endoplasmic Reticulum Extraction Kit (Solarbio, EX1370) and the Lysosome Enrichment Kit (Thermo Scientific, 89839), respectively. Briefly, cells were homogenized using a Dounce homogenizer, followed by ER enrichment through differential centrifugation and lysosome purification via density gradient separation according to the manufacturers’ instructions. All procedures were performed at 4°C with pre-cooled reagents.

Immunoprecipitation
For immunoprecipitation of V0a1 or Rab14 from RAW264.7 cells, cells were lysed with the RIPA lysis buffer supplemented with 1% protease inhibitor cocktail at 4°C for 10 minutes. Then, cell lysates were incubated with the antibody against V0a1 or Rab14 and protein A/G agarose (Santa Cruz Biotechnology, sc-2003) at 4°C, followed by washing with the RIPA lysis buffer for three times. For co-immunoprecipitation of two proteins co-overexpressed in HEK293T cells, cells were lysed in the RIPA lysis buffer at 4°C and were then incubated with anti-Flag M2 Affinity Gel (Sigma-Aldrich, A2220), anti-Myc Agarose (Santa Cruz Biotechnology, sc-40AC), or anti-GFP-Nanoab-Agarose (LABLEAD, GNA-50-1000) at 4°C for 4 hours. Thereafter, the cells were washed with the RIPA lysis buffer for three times. The immunoprecipitated samples were analyzed by immunoblotting with the indicated antibodies.

In vitro precipitation assay
For competitive binding analysis, 5 μg of His-CAMK2D was incubated with 20 μL of Ni-NTA Agarose in 500 μL binding buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM DTT, and 0.1% NP-40) containing 1% protease inhibitor cocktail for 1 hour at 4°C. Then, the agaroses carrying His-CAMK2D were collected and washed three times with the binding buffer, followed by incubating with 13 μg of purified GFP-V0a1 in the presence or absence of 1μg, 2 μg of GST or 2 μg, 5 μg of GST-Rab14 in the binding buffer for 4 hours at 4°C. After three washes, the bound protein complexes were finally subjected to immunoblotting analysis.

Acyl-Rac assay
The acyl-Rac assay was conducted to determine the S-palmitoylation of proteins as described previously60, 61. Briefly, proteins from cell lysates were incubated with blocking buffer (100 mM HEPES, 1 mM EDTA, 2.5 % SDS, 50 mM N- ethylmaleimide, pH 7.5) at 50℃ for 1 hour, followed by precipitation with three volumes of ice-cold acetone at -20℃ for 20 minutes. After centrifugation at 5,000 ×g for 10 minutes, the pellet was washed four times with 70% acetone. Next, the pellet was resuspended in binding buffer (100 mM HEPES, 1.0 mM EDTA, 1 % SDS, pH 7.5) and split into two aliquots. One aliquot was treated with 2 M NH2OH (freshly prepared, pH 7.5) to specifically cleave thioester bonds and expose nascent thiol groups, while the other was treated with PBS as a negative control. The newly exposed thiols were then biotinylated using EZ-Link™ HPDP-Biotin (Thermo Scientific, A35390). Biotinylated proteins were affinity-purified with Pierce™ streptavidin coated magnetic beads (Thermo Scientific, 89891) at room temperature for 2 hours. After three washes, the beads were mixed with loading buffer and subjected to immunoblotting analysis.

Immunoblotting analysis
Proteins were separated by SDS-PAGE gels and transferred to the polyvinylidene fluoride (PVDF) membrane (Millipore, IPVH00010). The membranes were blocked with 5% (w/v) nonfat dry milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) solution for 1 hour at room temperature and were then incubated with specific primary antibodies overnight at 4°C. Thereafter, the membranes were washed with TBST for three times and were then incubated with secondary antibodies for 1 hour at room temperature followed by three washes with TBST. Finally, the membranes were developed by Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500) and exposed to X-ray film. For quantifications, densitometry was performed using ImageJ (v1.8.0) to evaluate the intensity of the immunoblotting band signals. The uncropped and unprocessed scans of all blots are provided in the Source Data file.

Mass spectrometry (MS) analysis
For identifying Rab14-interacting proteins by MS analysis, immunoprecipitates of Flag or Flag-Rab14 were separated by SDS-PAGE. Then, the gels were stained with the Coomassie brilliant blue and the indicated bands were excised. Thereafter, the bands were rinsed with 100 mM ammonium bicarbonate, dried, and reduced using 10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate at 56°C and were then alkylated with 55 mM iodoacetamide. Proteins were digested with 12.5 ng/μL trypsin overnight at room temperature, and the peptide mixture was extracted by 60% acetonitrile. Next, samples were desalted on a C18 ZipTip pipette tip (Millipore) and were separated via Nano LC-MS/MS analysis on a NanoAcquity system (Waters, Milford, MA) connected to an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The MS raw files were processed using Proteome Discoverer (Version 1.4.0.288, Thermo Fischer Scientific) and were then searched against the Uniprot_human_2018 database by using the SEQUEST engine. The differential proteins with at least five unique peptides in the Flag-Rab14 group compared with the Flag group were considered the potential interactive proteins of Rab14, and the protein-protein interaction network diagram of Rab14-interacting proteins was edited by utilizing Adobe Illustrator CS6 (AI) software.

Immunofluorescence microscopy
Cells were seeded on cover glasses in 6-well cell culture plates and were then transfected with the indicated plasmids or infected with the indicated pathogens. Specifically, for detecting the colocalization of proteins with subcellular organelles, GFP-V0a1, GFP-V0a2, GFP-V0a3, or GFP-LC3 was transfected to Hela cells for 24 hours. Then, cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.5% Triton X-100 (Solarbio, T8200) for 15 minutes, and blocked with 5% BSA (Solarbio, A8010) for 30 minutes at room temperature. Primary antibodies against subcellular organelles including ER (anti-calnexin), Golgi (anti-GM130), early endosomes (anti-Rab5), late endosomes (anti-Rab7), and lysosomes (anti-LAMP1) were incubated with the cells for 2 hours at room temperature. After washing three times with 1 × PBS, the fluorophore-conjugated secondary antibodies were applied for 1 hour. After washing again, slides were mounted with medium containing 4′,6-diamidino-2-phenylindole (DAPI, Beyotime, C1002). For detecting lysosomal acidification during pathogen infections, RAW264.7 cells were infected with the indicated pathogen as described above and were then incubated with 75 nM LysoTracker for an additional 30 minutes. Next, cells were fixed and stained with DAPI. For detecting autophagy flux during pathogen infections, RAW264.7 cells stably expressing mCherry-GFP-LC3 were infected with the indicated pathogen as described above and were then fixed with 4% paraformaldehyde. For microscopy analysis, confocal images were taken with the Leica SP8 confocal microscope (Leica Microsystems) and analyzed by the Leica Application Suite Las X (v2.0.1.14392) software. For the image quantifications, approximately 50 cells were analyzed with ImageJ (v1.8.0) for each biological replicate, and the data was shown as the mean ± SEM of three independent experiments.

Live-cell imaging
For detecting V0a1 subcellular localization, Rab14+/+ and Rab14-/- RAW264.7 cells were plated on Glass Bottom Culture Dishes (NEST, 801002) and were then transfected with GFP-V0a1 for 24 hours. Thereafter, cells were infected with M. smegmatis prestained with Alexa FlourTM405 (Invitrogen, A30000) at a MOI of 10 for 2 hours and were then incubated with 75 nM LysoTracker for 30 minutes. Next, imaging was performed using a Leica SP8 confocal microscope equipped with an environmental control chamber providing 37°C, 5% CO2, and 20–30% humidity. Images were acquired in 5 minutes intervals over a time frame of 4 hours. For examining lysosomal acidification, the LysoSensor Yellow/Blue probe was added at 2 μM to cells and incubated at 37°C for 30 minutes. After being washed twice with pre-warmed growth medium, the cells were immediately visualized using a Leica SP8 confocal microscope. The fluorescence intensities in the blue and yellow channels were quantified using ImageJ (v1.8.0).

Molecular docking and multiple sequence alignment analysis
Molecular docking analysis of V0a1 and CAMK2D was performed as described previously62. Briefly, the structure of V0a1 (AlphaFold DB: AF-Q93050-F1) was predicted using AlphaFold 3, and the solved crystal structure of CAMK2D (PDB: 5VLO) was obtained from the RCSB PDB database. Both structures were then prepared by removing water molecules, heteroatoms, and co-crystallized ligands, followed by the addition of polar hydrogen atoms. The protein–protein molecular docking of V0a1 and CAMK2D was performed by ClusPro web server (https://cluspro.org). Interface analysis was performed using the PISA web server (https://www.ebi.ac.uk/pdbe/pisa/). Visualization and superimposition of docking poses and crystallographic structures were done using PyMOL (v.2.5.7). Additionally, multiple sequence alignment of V0a1, V0a2, and V0a3 was performed using ESPript 3.0 server (https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Conserved residues are marked with the red background.

In vitro and in vivo phosphorylation assay
In vitro phosphorylation assay detecting CAMK2D-mediated V0a1 phosphorylation was performed as described previously63. Briefly, 13 μg purified GFP-V0a1 or its mutants was incubated with 5 μg His-CAMK2D in the presence or absence of 5 μg GST-Rab14 in 200 μL kinase reaction buffer (10 mM HEPES, pH 7.2, 1 mM EGTA, 5 mM MgCl2, 2 mM CaCl2, and 0.4 mM ATP) supplemented with 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail. After incubation at 30°C for 30 minutes, the reaction was terminated by the addition of SDS loading buffer and subjected to SDS-PAGE analysis. V0a2 and V0a3 phosphorylation was examined using the same method. For in vivo phosphorylation assay, V0a1 or its mutant was immunoprecipitated from Rab14+/+ and Rab14-/- cells and was then subjected to immunoblotting with the antibody against p-Ser or p-Thr.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 software (https://www.graphpad.com/). All data were analyzed using unpaired two-sided Student’s t-test, one-way ANOVA, or two-way ANOVA analysis followed by multiple comparisons as indicated in the corresponding figure legends. Data are shown as mean and standard error of mean (s.e.m.). Unless otherwise indicated, at least two biological replicates were included in all experiments. Additional details about the statistical analysis of experiments and number of biological replicates (n) are indicated in the corresponding figure legends.

Data availability
All data are available in the main text and the supplementary materials. The plasmids and cell lines generated in this study are available from the corresponding author. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The host-directed restriction factors against viral infections are well studied. However, such factors against bacterial\npathogens largely remain unexplored. Lei and colleagues in their manuscript (NCOMMS-25-55033-T) entitled “Rab14\nrestricts pathogens by promoting V-ATPase lysosomal delivery to drive lysosomal acidification” conducted a comprehensive\nscreening of host factors that promote lysosomal acidification, which is essential for pathogen clearance from infected cells.\nThey screened host factors that promote lysosomal acidification, and identified a small GTPase, Rab14, as a restriction\nfactor with broad-spectrum activity against multiple viruses and bacteria. Since\nlysosomal acidification is essential for lysosomal acid hydrolases to degrade the\npathogens, they focused their investigations on understanding the mechanism\nunderlying the events that follow an infection. They found that upon infection by a pathogen, the level of GTP-bound Rab14\nbinding to Calcium/calmodulin-dependent protein kinase 2 (CAMK2D) increases. This process suppressed CAMK2D-\nmediated phosphorylation of V0a1, a critical subunit essential for the assembly of the multi-protein complex representing\nfunctional V-ATPase, which acidifies the lysosomal lumen. The results of their study have uncovered a previously\nunrecognized intrinsic mechanism that regulates the innate immune response that protects an organism against infection by\nbacterial pathogens.\nGeneral comments-\nThis is an imaginative, well-designed and executed study that provides a considerable amount of novel information that may\nbe useful for the development of effective therapeutics against antibiotic-resistant bacterial pathogens. Having said that,\nsome of the results of this study may require further elaboration to fully explain the proposed mechanisms. Therefore, the\nmanuscript may require some major revisions.\nSpecific comments:\n1.\nPrevious mechanistic studies have shown that Rab14 stimulates organellar fusion (e.g. phagosomes-early endosome) but\nnot between phagosome and late endosome/lysosome. Moreover, Rab14 has been reported to enable phagosomes\ncontaining tuberculosis bacteria to maintain early endosomal characteristics and thereby, resist the progression to late\nendosomal / lysosomal degradative functon(https://doi:10.1038/sj.emboj.7601407). Since autophagosome-lysosome fusion\nis critical for the catalytic function of the lysosomal acid hydrolases that are essential for degrading the contents of the\nautophagosomes. The authors should also discuss how Rab14-CAMK2D-mediated phosphorylation of V0a1 may\ncoordinately regulate the two processes (i.e. the localization of V0a1 on lysosomal membrane to maintain lysosomal\nacidification by VATPase and the autophagosome-lysosome fusion).\n2.\nThe authors highlighted the critical roles of Rab14 in host-directed restriction against pathogens. However, recent reports\nindicate that both Rab2 and Rab14 are important in autophagosome maturation and function requiring lysosomal\nacidic environment (https://doi.org/10.1080/15548627.2024.2374699), which is maintained by active V-ATPase on\nlysosomal membrane. The authors should discuss whether Rab2, in addition to Rab14, plays any role in lysosomal\nacidification in their experimental system.\n3.\nOn page 7/79, lines 152-156, the authors state that while V0a1 strongly colocalized with LAMP1-positive lysosomes in\nRab14+/+ cells, it primarily colocalized with the ER-marker, calnexin in Rab14-/- cells. The anterograde movement (from ER\nto Golgi) of proteins is mediated by COPII vesicles. In addition, all COPII proteins, except Sec13, have been reported to\nundergo S-palmitoylation (https://doi:10.1016/j.nbd.2025.106890), a reversable post-translational modification of proteins by\nsaturated fatty acids (generally palmitate). Moreover, dynamic S-palmitoylation (palmitoylation-depalmitoylation) has been\nshown to facilitate the trafficking of V0a1 to the late endosome / lysosome (https://doi:10.1038/ncomms14612). Thus, both S-\npalmitoylation and depalmitoylation, catalyzed by ZDHHC palmitoyl acyl transferases (PATs) and thioesterases,\nrespectively, are critical for V0a1 to successfully traffic to the late endosomal / lysosomal membrane\n(https://doi:10.1098/rsob.200411). Indeed, it has been reported that the handover of V0a1 from the sorting endosome to late\nendosome / lysosome requires dynamic S-palmitoylation. Therefore, it is critically important for the authors to rule out that in\nRab14-/- cells, the dynamic S-palmitoylation of V0a1 is not compromised impairing the assembly of VATPase multiprotein\ncomplex essential for trafficking from the ER to the lysosomal membrane.\n4.\nThe authors have demonstrated that in Rab14-/- cells V0a1 colocalizes with the ER marker, calnexin. This may suggest that\nthe ER-Golgi trafficking is also defective in these cells. Since COPII proteins (e.g. Sar1, Sec23/Sec24) except Sec 13 are\nalso reported to be S-palmitoylated (https://doi: 10.1016/j.nbd.2025.106890), and a thioesterase (PPT1)-deficiency impairs\nanterograde (ER to Golgi) trafficking mediated by COPII vesicles, it is therefore critically important for the authors to\ndemonstrate that dynamic S-palmitoylation of COPII-associated proteins are not adversely affected in Rab14-/- cells.\n5.\nA role for Rab14 in stimulating the fusion of phagosomes with early endosomes but not with late endosomes has been\ndemonstrated in mechanistic studies (https://doi:10.1038/sj.emboj.7601407). In case of mycobacterial pathogens, it has\nbeen shown that Rab14 enables phagosomes to maintain early endosomal characteristics but not those of late\nendosomal/lysosomal degradative components. The authors should discuss this finding considering their proposed immune\nmechanism mediated by Rab14-CAM2D-V-ATPase axis.\nMinor comments:\n--Figure 1 panels g-j: The Western blot to demonstrate the processing of pro-cathepsin D (CD) to enzymatically active-CD\nappears to be unclear. The pro-CD (48 kDa) undergoes proteolytic cleavage in the lysosome by Cathepsin B and Cathepsin\nL, respectively, to generate the 31kDa and 14 kDa fragments. Non-covalent dimerization of the 31kDa and 14 kDa\nfragments, generates the enzymatically active-CD (https://doi.org/10.1016/j.bbrc.2008.08.099). The Western blots in Figs.\n1g-1j do not attest to this picture. It appears that the 14kDa band is missing in the blots. The use of higher percentage gels\nand longer duration of electrophoresis may resolve this problem.\n--A similar problem is encountered in Figure 6b-e. The blots should clearly show the presence of the 31kDa and 14kDa\nbands. An alternative may be to determine the CD enzymatic activity in Rab+/+ and Rab-/- cells. The Rab14-/- cells should\nshow significantly reduced CD enzymatic activity compared with that in Rab14+/+ cells.", "reviewer_2": "The authors identified Rab14 as a restriction factor against multiple bacteria and viruses. They showed that Rab14 binds to\nand inhibits CAMK2D-mediated phosphorylation of V0a1 and thus promotes lysosome acidification for pathogen clearance.\nThe finding of RAB14 in V0a1 trafficking is interesting. Both imaging data and quantification analyses need to be improved\nsubstantially to support the conclusions.\nMajor points:\n1. Fluorescence images in all figures are too small to see clear subcellular localization and the alterations under different\nconditions. High magnification images showing clear subcellular compartments (ER, Golgi, lysosomes) and the localization\nof the target proteins (V0a) should be provided.\n2. Quantification analyses are lacking in many assays.\nSpecific comments:\n1. Lysotracker staining in Fig. 1b-f should be done in cells labeled by lysosome membrane markers such as LAMP1 or\nTMEM192, to confirm defects in acidification but not number of lysosomes.\n2. Fig. 1g-i: CTSD processing should be quantified.\nExtended Fig. 2: LC3 processing should be quantified.\n3. Data shown in Fig. 2a-e, f are critical, but the images are unclear. High magnification images are needed to indicate\nsubcellular localization of V0a1 with different markers.\nIn Fig. 2f, lysosomal marker such as LAMP1 should be included. Lysotracker staining shows lysosomal acidification, but it is\nnot a suitable lysosomal marker to show V01a trafficking to lysosomes in these experiments.\n4. Why V0a1, but not V0a2 and a3, is affected in Rab14-deficient cells? As all of them are expressed in macrophages, how\nthe specific regulation of V0a1 by Rab14 is achieved?\n5. Quantification analyses should be provided in Fig. 3a-d; high magnification and more clear images are needed in Fig. 3e,\nf.\n6. Extended Fig. 5e, CAMK2D interacts with V0a1 in both Rab14+/+ and Rab14-/- cells, which is inconsistent with the\nstatement in the manuscript (line 209-210). Quantification analyses should be included to support the conclusion.\n7. The p-Ser specificity should be examined.\n8. Where CAMK2D is localized and whether its localization is altered in Rab14-/- cells?\n9. Quantification analyses should be provided in Fig. 5a-h, and high magnification and more clear images are needed in Fig.\n5i.\n10. Quantification analyses should be provided in Fig. 6b-e,", "reviewer_3": "This article provides insights into how Rab14 contributes to pathogen clearance by regulating the CAMK2D-ATPase axis.\nOverall, the data are informative and support the main conclusions. I have several comments that may help strengthen the\nmanuscript:\n1. Recent work has shown that RAB14, together with other proteins, regulates autophagy by modulating the HOPS complex.\nThis suggests that RAB14 may influence autophagy independently of lysosomal acidification. In Extended Data Fig. 8, it\nappears that the maturation of LC3 puncta during autophagy is entirely regulated by the Rab14-CAMK2D axis. The authors\nmay need to further discuss this point in the discussion. It would be also good to explain more by new data if it's necessary.\n2. In Fig. 2, the conclusions rely mainly on imaging analysis. It would be valuable to include complementary approaches to\nsupport the findings. In addition, some of the representative images do not adequately reflect the statistical analysis.\nSelecting clearer images would strengthen the presentation. Furthermore, about 50 cells were analyzed per biological\nreplicate. Plotting the ratios from each individual cell would make the data more convincing.\n3. For Fig. 3e, f, the same concern applies: some of the representative images are not optimal and could be replaced with\nmore suitable ones to better reflect the quantitative analysis.\n4. In Fig. 5i, the authors conclude that “Rab14 promotes the trafficking of V0a1 to lysosomes in a manner dependent on\nCAMK2D during pathogen infections.” However, the data indicate that Rab14 also promotes V0a1 trafficking to lysosomes\nunder non-infection conditions. The conclusion should therefore be revised accordingly.\n5. LysoTracker (Invitrogen, L7528) is not a direct probe for intracellular pH. To validate the key findings, the authors are\nencouraged to repeat some key experiments using LysoSensor probes, which are specifically designed for pH\nmeasurement.\nMinor points:\n1. Line 94: The reference to “Supplementary Fig. 1” may need to be deleted.\n2. Extended Data Fig. 2b: It would improve clarity if the long-term and short-term LC3 imaging results were presented\ntogether."}, "author_response": "Point-by-point responses to reviewers’ comments\nRE: Manuscript (NCOMMS-25-55033-T) “Rab14 restricts pathogens by\npromoting V-ATPase lysosomal delivery to drive lysosomal\nacidification” by Lei et al.\nReviewers’ Comments:\nReviewer #1:\nThe host-directed restriction factors against viral infections are well studied. However,\nsuch factors against bacterial pathogens largely remain unexplored. Lei and colleagues\nin their manuscript (NCOMMS-25-55033-T) entitled “Rab14 restricts pathogens by\npromoting V-ATPase lysosomal delivery to drive lysosomal acidification” conducted a\ncomprehensive screening of host factors that promote lysosomal acidification, which is\nessential for pathogen clearance from infected cells. They screened host factors that\npromote lysosomal acidification, and identified a small GTPase, Rab14, as a restriction\nfactor with broad-spectrum activity against multiple viruses and bacteria. Since\nlysosomal acidification is essential for lysosomal acid hydrolases to degrade the\npathogens, they focused their investigations on understanding the mechanism\nunderlying the events that follow an infection. They found that upon infection by a\npathogen, the level of GTP-bound Rab14 binding to Calcium/calmodulin-dependent\nprotein kinase 2 (CAMK2D) increases. This process suppressed CAMK2D-mediated\nphosphorylation of V0a1, a critical subunit essential for the assembly of the multi-\nprotein complex representing functional V-ATPase, which acidifies the lysosomal\nlumen. The results of their study have uncovered a previously unrecognized intrinsic\nmechanism that regulates the innate immune response that protects an organism against\ninfection by bacterial pathogens.\nGeneral comments-\nThis is an imaginative, well-designed and executed study that provides a considerable\namount of novel information that may be useful for the development of effective\ntherapeutics against antibiotic-resistant bacterial pathogens. Having said that, some of\nthe results of this study may require further elaboration to fully explain the proposed\nmechanisms. Therefore, the manuscript may require some major revisions.\nR: We thank the reviewer for the insightful comments and valuable suggestions.\nWe have improved the manuscript by performing additional experiments and\nexpanding the discussion. The following is the point-by-point response to all\ncomments.\nSpecific comments:\n1. Previous mechanistic studies have shown that Rab14 stimulates organellar fusion\n(e.g. phagosomes-early endosome) but not between phagosome and late\nendosome/lysosome. Moreover, Rab14 has been reported to enable phagosomes\ncontaining tuberculosis bacteria to maintain early endosomal characteristics and\nthereby, resist the progression to late endosomal / lysosomal degradative function\n(https://doi:10.1038/sj.emboj.7601407). Since autophagosome-lysosome fusion is\ncritical for the catalytic function of the lysosomal acid hydrolases that are essential for\ndegrading the contents of the autophagosomes. The authors should also discuss how\nRab14-CAMK2D-mediated phosphorylation of V0a1 may coordinately regulate the\ntwo processes (i.e. the localization of V0a1 on lysosomal membrane to maintain\nlysosomal acidification by V-ATPase and the autophagosome-lysosome fusion).\nR: We thank the reviewer for raising this critical point. As noted by the reviewer,\nRab14 has been reported to promote phagosome-early endosome fusion while\nsuppressing phagosome fusion with late endosomes or lysosomes (PMID:\n17082769). However, it should be mentioned that this function was characterized\nin macrophages infected with Mycobacterium tuberculosis (Mtb), a chronic\npathogen that usually hijacks host factors to inhibit immune responses for its long-\nterm survival (PMID: 32728204). For example, Mtb can employ its effectors (such\nas PknG) to suppress Rab14-GTP hydrolysis, leading to the constitutive activation\nof Rab14, which arrests phagosomes at the early endosome stage and prevents\ntheir fusion with late endosomes and lysosomes, thereby blocking phagosomal\nmaturation and pathogen clearance (PMID: 17082769; PMID: 34092182). In\ncontrast, in macrophages infected with heat-killed Mtb, Rab14 only transiently\nassociates with phagosomes, permitting their normal fusion with late endosomes\nand lysosomes for bacterial clearance (PMID: 17082769). Thus, the previously\nobserved effect of Rab14 stimulating organellar fusion between phagosomes and\nearly endosomes, but not with late endosomes or lysosomes, may represent a\npathogen-hijacked state. In our study, instead of using Mtb that hijacks Rab14\nfunction, we used model pathogens (including Mycobacterium smegmatis, Listeria\nmonocytogenes, DNA virus HSV-1, and RNA virus SeV) that can be rapidly cleared\nby the host immune system to investigate the uncompromised, physiological\nfunction of Rab14, and we demonstrate that Rab14 primarily facilitates pathogen\nclearance by promoting V0a1 lysosomal localization and subsequent lysosomal\nacidification through suppression of CAMK2D-mediated V0a1 phosphorylation.\nFurthermore, we performed confocal microscopy experiments to address the\nreviewer’s query regarding whether the Rab14-CAMK2D axis modulates the\nautophagosome-lysosome fusion step, and we found that the Rab14-CAMK2D axis\ndid not regulate this process in macrophages (revised Supplementary Fig. 13; lines\n375–384). Thus, Rab14-CAMK2D-mediated phosphorylation of V0a1 regulates\nthe localization of V0a1 on lysosomal membrane to maintain lysosomal\nacidification by V-ATPase without affecting the autophagosome-lysosome fusion.\nIt should be noticed that our macrophage-based results are different from a\nprevious study reporting that Rab14 promotes autophagosome-lysosome fusion in\nMDCK epithelial cells (PMID: 38953305), indicating that the function of Rab14 is\ncell type-dependent, which is further supported in other studies. For instance,\nRab14 promotes the trafficking of glucose transporter 4 (GLUT4) from early\nendosomes to the Golgi apparatus following endocytosis in adipocytes (PMID:\n23444368; PMID: 26936971), while facilitating the GLUT4 translocation from the\nGLUT4 storage vesicles (GSV) compartment to the plasma membrane in muscle\ncells (PMID: 18701652).\nTaken together, Rab14 is a critical host restriction factor whose activity is fine-\ntuned by cell-type-specific mechanisms, which process could also be exploited by\ncertain pathogens for their immune evasion. We have expanded our discussion\naccordingly (lines 433–434, 440–451).\n2. The authors highlighted the critical roles of Rab14 in host-directed restriction against\npathogens. However, recent reports indicate that both Rab2 and Rab14 are important in\nautophagosome maturation and function requiring lysosomal acidic environment\n(https://doi.org/10.1080/15548627.2024.2374699), which is maintained by active V-\nATPase on lysosomal membrane. The authors should discuss whether Rab2, in addition\nto Rab14, plays any role in lysosomal acidification in their experimental system.\nR: We thank the reviewer for raising this question. A recent study demonstrated\nthat both Rab2 and Rab14 facilitate the autophagosome maturation through\nrecruiting the HOPS complex to promote autophagosome-lysosome fusion (PMID:\n38953305). Notably, this fusion step is independent of V-ATPase-mediated\nlysosomal acidification (PMID: 25959678; PMID: 26156798); thus, the role of\nRab2 and Rab14 in fusion does not imply a function in modulating lysosomal pH.\nIn our experimental system, lysosomal pH analysis was conducted in macrophages\nwith Rab gene knockdown, the data showed that unlike Rab14 knockdown, Rab2a\nknockdown minimally altered lysosomal pH (Fig. 1a). Thus, Rab14 exerts a\nspecific function in promoting lysosomal acidification, which is not shared by\nRab2 (lines 102–105).\n3. On page 7/79, lines 152-156, the authors state that while V0a1 strongly colocalized\nwith LAMP1-positive lysosomes in Rab14+/+ cells, it primarily colocalized with the\nER-marker, calnexin in Rab14-/- cells. The anterograde movement (from ER to Golgi)\nof proteins is mediated by COPII vesicles. In addition, all COPII proteins, except Sec13,\nhave been reported to undergo S-palmitoylation (:10.1016/j.nbd.2025.106890), a\nreversable post-translational modification of proteins by saturated fatty acids (generally\npalmitate). Moreover, dynamic S-palmitoylation (palmitoylation-depalmitoylation) has\nbeen shown to facilitate the trafficking of V0a1 to the late endosome / lysosome\n(https://doi:10.1038/ncomms14612). Thus, both S-palmitoylation and depalmitoylation,\ncatalyzed by ZDHHC palmitoyl acyl transferases (PATs) and thioesterases, respectively,\nare critical for V0a1 to successfully traffic to the late endosomal / lysosomal membrane\n(https://doi:10.1098/rsob.200411). Indeed, it has been reported that the handover of\nV0a1 from the sorting endosome to late endosome / lysosome requires dynamic S-\npalmitoylation. Therefore, it is critically important for the authors to rule out that in\nRab14-/- cells, the dynamic S-palmitoylation of V0a1 is not compromised impairing the\nassembly of VATPase multiprotein complex essential for trafficking from the ER to the\nlysosomal membrane.\nR: We thank the reviewer for this valuable suggestion. As advised, we performed\nacyl-Rac assay to determine the S-palmitoylation of V0a1 in Rab14+/+ and Rab14-\n/- cells, and we observed no significant difference in V0a1 palmitoylation levels\nbetween Rab14+/+ and Rab14-/- cells (revised Supplementary Fig. 7b; lines 217–219,\n777–792). Moreover, the S-palmitoylation of V0a1 is required for its trafficking\nfrom the sorting endosome to the late endosomal/lysosomal membrane (PMID:\n28266544), while Rab14 mainly modulates the delivery of V0a1 from the ER to\nGolgi, which is a different trafficking way independent of the endosomal sorting\npathway. Taken together, Rab14 promotes V0a1 trafficking from the ER to the\nlysosomal membrane independent of V0a1 S-palmitoylation.\n4. The authors have demonstrated that in Rab14-/- cells V0a1 colocalizes with the ER\nmarker, calnexin. This may suggest that the ER-Golgi trafficking is also defective in\nthese cells. Since COPII proteins (e.g. Sar1, Sec23/Sec24) except Sec 13 are also\nreported to be S-palmitoylated (https://doi: 10.1016/j.nbd.2025.106890), and a\nthioesterase (PPT1)-deficiency impairs anterograde (ER to Golgi) trafficking mediated\nby COPII vesicles, it is therefore critically important for the authors to demonstrate that\ndynamic S-palmitoylation of COPII-associated proteins are not adversely affected in\nRab14-/- cells.\nR: We thank the reviewer for this valuable suggestion. We conducted acyl-Rac\nassay to determine the S-palmitoylation of COPII-associated proteins including\nSar1, Sec23A, Sec24A, Sec24C, and Sec31A in Rab14+/+ and Rab14-/- cells, and we\nobserved comparable S-palmitoylation levels of these proteins in Rab14+/+ and\nRab14-/- cells (revised Supplementary Fig. 9b; lines 273–278, 777–792), indicating\nthat Rab14 is not involved in regulating COPII complex S-palmitoylation.\n5. A role for Rab14 in stimulating the fusion of phagosomes with early endosomes but\nnot with late endosomes has been demonstrated in mechanistic studies\n(https://doi:10.1038/sj.emboj.7601407). In case of mycobacterial pathogens, it has\nbeen shown that Rab14 enables phagosomes to maintain early endosomal characteristics but\nnot those of late endosomal/lysosomal degradative components. The authors should\ndiscuss this finding considering their proposed immune mechanism mediated by\nRab14-CAM2D-V-ATPase axis.\nR: We thank the reviewer for raising this critical point. As previously explained in\nour response to question #1, Rab14 has been reported to promote phagosome-early\nendosome fusion while suppressing phagosome fusion with late endosomes or\nlysosomes in macrophages infected with Mtb (PMID: 17082769). It should be\npointed out that Mtb is a chronic pathogen that usually hijacks host factors to\ninhibit immune responses for its long-term survival (PMID: 32728204). For\nexample, Mtb can employ its effectors (such as PknG) to suppress Rab14-GTP\nhydrolysis, leading to the constitutive activation of Rab14, which arrests\nphagosomes at the early endosome stage and prevents their fusion with late\nendosomes and lysosomes, thereby blocking phagosomal maturation and\npathogen clearance (PMID: 17082769; PMID: 34092182). In contrast, in\nmacrophages infected with heat-killed Mtb, Rab14 only transiently associates with\nphagosomes, permitting their normal fusion with late endosomes and lysosomes\nfor bacterial clearance (PMID: 17082769). Thus, the previously observed effect of\nRab14 stimulating organellar fusion between phagosomes and early endosomes,\nbut not with late endosomes or lysosomes, may represent a pathogen-hijacked\nstate. In our study, instead of using Mtb that hijacks Rab14 function, we used\nmodel pathogens (including Mycobacterium smegmatis, Listeria monocytogenes,\nDNA virus HSV-1, and RNA virus SeV) that can be rapidly cleared by the host\nimmune system to investigate the uncompromised, physiological function of\nRab14, and we demonstrate that Rab14 primarily facilitates pathogen clearance\nby promoting V0a1 lysosomal localization and subsequent lysosomal acidification\nthrough suppression of CAMK2D-mediated V0a1 phosphorylation. Thus, Rab14\nis a critical host restriction factor whose activity could be exploited by certain\npathogens for their immune evasion. We have expanded our discussion\naccordingly (lines 433–434, 440–444, 448–451).\nMinor comments:\n1. Figure 1 panels g-j: The Western blot to demonstrate the processing of pro-cathepsin\nD (CD) to enzymatically active-CD appears to be unclear. The pro-CD (48 kDa)\nundergoes proteolytic cleavage in the lysosome by Cathepsin B and Cathepsin L,\nrespectively, to generate the 31kDa and 14 kDa fragments. Non-covalent dimerization\nof the 31kDa and 14 kDa fragments, generates the enzymatically active-CD\n(https://doi.org/10.1016/j.bbrc.2008.08.099). The Western blots in Figs. 1g-1j do not\nattest to this picture. It appears that the 14kDa band is missing in the blots. The use of\nhigher percentage gels and longer duration of electrophoresis may resolve this problem.\nA similar problem is encountered in Figure 6b-e. The blots should clearly show the\npresence of the 31kDa and 14kDa bands. An alternative may be to determine the CD\nenzymatic activity in Rab+/+ and Rab-/- cells. The Rab14-/- cells should show\nsignificantly reduced CD enzymatic activity compared with that in Rab14+/+ cells.\nR: We thank the reviewer for raising this important question and for the helpful\nsuggestions. In our experiments, we used two distinct antibodies targeting the\nheavy chain (31 kDa) and light chain (14 kDa) of mature CTSD, respectively. Our\nprevious manuscript presented only the heavy chain data, in line with common\npractice reported in other studies (e.g., PMID: 35287553; PMID: 31282275). In\nresponse to the reviewer’s comment, we now provide the corresponding light chain\ndata derived from the same samples (revised Fig. 1e–h and Fig. 6b–e), which\nsimilarly demonstrates that Rab14 promotes CTSD maturation. Furthermore, as\nsuggested by the reviewer, we additionally performed the CTSD activity assay and\nconfirmed that the Rab14-CAMK2D axis enhances CTSD proteolytic activity\n(revised Supplementary Fig. 3c and Supplementary Fig. 11a; lines 131–134, 364–\n367, 722–728).\nReviewer #2:\nThe authors identified Rab14 as a restriction factor against multiple bacteria and viruses.\nThey showed that Rab14 binds to and inhibits CAMK2D-mediated phosphorylation of\nV0a1 and thus promotes lysosome acidification for pathogen clearance. The finding of\nRAB14 in V0a1 trafficking is interesting. Both imaging data and quantification analyses\nneed to be improved substantially to support the conclusions.\nR: We thank the reviewer for these constructive comments. We have improved our\nmanuscript by optimizing the imaging data and adding quantification results for\nthe immunoblots. The following is the point-by-point response to all comments.\nMajor points:\n1. Fluorescence images in all figures are too small to see clear subcellular localization\nand the alterations under different conditions. High magnification images showing clear\nsubcellular compartments (ER, Golgi, lysosomes) and the localization of the target\nproteins (V0a) should be provided.\nR: We thank the reviewer for the valuable suggestion. We repeated the confocal\nmicroscopy experiments with optimized conditions and provided higher\nmagnification and clearer images in our revised manuscript (revised Fig. 2, Fig.\n3e, f, and Fig. 5i).\n2. Quantification analyses are lacking in many assays.\nR: We thank the reviewer for pointing out this issue. We performed quantification\nanalyses for the immunoblots and included these data in the revised manuscript\n(revised Fig. 1e–h, Fig. 3a–d, Fig. 5a–h, Fig. 6b–e, Supplementary Fig. 4a–d, and\nSupplementary Fig. 8d).\nSpecific comments:\n1. Lysotracker staining in Fig. 1b-f should be done in cells labeled by lysosome\nmembrane markers such as LAMP1 or TMEM192, to confirm defects in acidification\nbut not number of lysosomes.\nR: We thank the reviewer for this valuable suggestion. To definitively determine\nwhether the observed reduction in LysoTracker signal reflects impaired lysosomal\nacidification rather than a decrease in lysosome number, we performed the\nrecommended co-staining for LAMP1 (a marker of total lysosomes) and\nLysoTracker (a marker of acidified lysosomes). Our data showed that while the\nLysoTracker fluorescence intensity was much higher in infected Rab14+/+\nmacrophages than that in infected Rab14-/- macrophages, the LAMP1 intensity\nremained comparable between the two group (Fig. 1b–d; lines 112–118),\nconfirming that Rab14 depletion specifically compromises lysosomal acidification\nwithout affecting lysosomal number. To further validate this finding functionally,\nwe performed live-cell imaging assays using the LysoSensor Yellow/Blue probe,\nwhich mainly produces yellow fluorescence in acidic lysosomes (low pH), but blue\nfluorescence in less acidic lysosomes (high pH) (PMID: 11444806). The results\nconsistently confirmed that Rab14 promotes lysosomal acidification\n(Supplementary Fig. 3b; lines 118–124, 861–866). Together, these results from\nexperiments adopting two approaches conclusively demonstrate that Rab14 is\nrequired for lysosomal acidification, independent of lysosomal biogenesis.\n2. Fig. 1g-i: CTSD processing should be quantified.\nExtended Fig. 2: LC3 processing should be quantified.\nR: We thank the reviewer for this helpful suggestion. We performed quantification\nanalyses for CTSD maturation (calculated by the ratio of mature CTSD heavy\nchain or light chain to TUBA1A, revised Fig. 1e–h) and LC3 processing (calculated\nby the LC3-II/TUBA1A ratio, revised Supplementary Fig. 4a–d). The\nquantification results provide statistical validation and consistently demonstrate\nthat Rab14 is required for the efficient processing of both proteins.\n3. Data shown in Fig. 2a-e, f are critical, but the images are unclear. High magnification\nimages are needed to indicate subcellular localization of V0a1 with different markers.\nIn Fig. 2f, lysosomal marker such as LAMP1 should be included. Lysotracker staining\nshows lysosomal acidification, but it is not a suitable lysosomal marker to show V01a\ntrafficking to lysosomes in these experiments.\nR: We sincerely thank the reviewer for pointing out these issues. We repeated the\nconfocal microscopy experiments and optimized the imaging conditions to more\nclearly resolve the subcellular localization of V0a1. The double-\nimmunofluorescence labeling analysis of V0a1 and organelle markers revealed\nstrong colocalization of V0a1 with LAMP1-positive lysosomes in Rab14+/+ cells,\nwhereas in Rab14-/- cells, V0a1 primarily colocalized with the ER-integral protein\ncalnexin (revised Fig. 2a–e). Moreover, we isolated the ER and lysosomal fractions\nfrom Rab14⁺/⁺ and Rab14⁻/⁻ cells and consistently demonstrated that Rab14\npromotes the lysosomal targeting of V0a1 (revised Supplementary Fig. 5a; lines\n175–179, 745–751). Furthermore, regarding lysosomal markers, as suggested by\nthe reviewer, we used V0a1 and the canonical lysosomal membrane protein\nLAMP1 for co-immunostaining experiments, and the results showed that\npathogen infections could increase the trafficking of V0a1 to lysosomes in Rab14+/+\ncells, while this effect is largely reduced in Rab14-/- cells (revised Fig. 2f). Analysis\nof V0a1 levels in purified lysosomes also demonstrated a significantly higher\nenrichment of V0a1 on lysosomes from Rab14+/+ cells than those from Rab14-/- cells\n(revised Supplementary Fig. 6a; lines 189–191).\n4. Why V0a1, but not V0a2 and a3, is affected in Rab14-deficient cells? As all of them\nare expressed in macrophages, how the specific regulation of V0a1 by Rab14 is\nachieved?\nR: We thank the reviewer for raising this insightful question regarding the specific\nregulation of V0a1, but not V0a2 or V0a3, by Rab14 in macrophages. In our\nprevious manuscript, we found that Rab14 selectively promotes the lysosomal\ntrafficking of V0a1 without affecting V0a2 or V0a3, which led us to focus on V0a1\nto dissect the underlying mechanism. Mechanistical studies revealed that Rab14\nbinds to CAMK2D and competitively inhibits the interaction between CAMK2D\nand V0a1, thus suppressing CAMK2D-mediated phosphorylation of V0a1 to\npromote V0a1 binding to the COPⅡ complex for V0a1 trafficking from the ER to\nGolgi and then to lysosomes. Thus, the interaction between CAMK2D and V0a1\nrepresents the core mechanism by which Rab14 regulates V0a1 trafficking.\nTo further elucidate the mechanism by which Rab14 specifically regulates V0a1,\nas requested by the reviewer, we detected the interaction between CAMK2D and\nV0a1, V0a2, or V0a3, and we found that CAMK2D specifically bound to V0a1, but\nnot V0a2 or V0a3 (Supplementary Fig. 8a; lines 236–237). We further conducted\nmolecular docking analysis and revealed that CAMK2D bound to the Arg 179 and\nArg 198 sites of V0a1, two residues that are not conserved in V0a2 or V0a3\n(Supplementary Fig. 8b, c; lines 237–239, 868–881). Consistently, the in vitro\nphosphorylation assay showed that V0a1, but not V0a2 or V0a3, was\nphosphorylated by CAMK2D (Supplementary Fig. 8g; lines 248–249). Taken\ntogether, the specificity of Rab14 for V0a1 is determined by the exclusive,\nstructure-dependent interaction between CAMK2D and V0a1.\n5. Quantification analyses should be provided in Fig. 3a-d; high magnification and more\nclear images are needed in Fig. 3e, f.\nR: We thank the reviewer for these helpful suggestions. We have provided\nquantification results for the immunoblots in the revised Fig. 3a–d, with the\ndetailed methodology described in the corresponding figure legends (lines 1149–\n1150, 1152–1153, 1155–1156, 1161–1162). Additionally, we repeated the confocal\nmicroscopy experiments and provided clearer, high-magnification images in the\nrevised Fig. 3e–f to better illustrate the subcellular localization of V0a1.\n6. Extended Fig. 5e, CAMK2D interacts with V0a1 in both Rab14+/+ and Rab14-/- cells,\nwhich is inconsistent with the statement in the manuscript (line 209-210).\nQuantification analyses should be included to support the conclusion.\nR: We thank the reviewer for raising this concern. We performed quantification\nanalyses for the co-immunoprecipitation experiments in the previous Extended\nData Fig. 5e. The quantitative results confirmed that the interaction between\nCAMK2D and V0a1 is indeed significantly stronger in Rab14-/- cells compared to\nthat in Rab14+/+ cells (revised Supplementary Fig. 8d), which is consistent with our\nstated conclusion that Rab14 competitively inhibits the binding between\nCAMK2D and V0a1.\n7. The p-Ser specificity should be examined.\nR: We thank the reviewer for raising this critical point regarding the p-Ser\nspecificity. In our experimental system for detecting the p-Ser of V0a1, we first\nimmunoprecipitated V0a1 followed by p-Ser immunoblotting. The critical\nevidence for the specificity is that the p-Ser antibody failed to detect any signal at\nthe expected molecular weight when using the V0a1S19A mutant, demonstrating\nthat the phospho-serine signal we observed was specifically derived from V0a1 and\ndependent on the S19 residue. Moreover, we generated a phosphospecific antibody\nagainst the V0a1 S19 site. Through in vitro and in vivo phosphorylation assays\nusing this antibody, we confirmed that CAMK2D phosphorylates V0a1 at S19,\nwhile Rab14 inhibits this process (Supplementary Fig. 9f, g; lines 299–303, 597–\n604).\n8. Where CAMK2D is localized and whether its localization is altered in Rab14-/- cells?\nR: We thank the reviewer for raising this question. Through examining the\nsubcellular localization of CAMK2D by confocal microscopy analysis, we found\nthat CAMK2D exhibits a diffuse cytoplasmic distribution, and this localization\npattern remains unchanged in Rab14-/- cells (Supplementary Fig. 9a; lines 266–\n270), indicating that Rab14 modulates V0a1 trafficking independent of CAMK2D\nlocalization.\n9. Quantification analyses should be provided in Fig. 5a-h, and high magnification and\nmore clear images are needed in Fig. 5i.\nR: We thank the reviewer for these helpful suggestions. We have provided\nquantification results for the immunoblots in the revised Fig. 5a–h, with the\ndetailed methodology described in the corresponding figure legends (lines 1205–\n1206, 1212). Additionally, we repeated the confocal microscopy experiments and\nprovided clearer, high-magnification images in the revised Fig. 5i to better\nillustrate the subcellular localization of V0a1.\n10. Quantification analyses should be provided in Fig. 6b-e.\nR: We thank the reviewer for this helpful suggestion. We have added\nquantification analyses for CTSD maturation, represented by the ratio of mature\nCTSD heavy chain or light chain to TUBA1A (revised Fig. 6b–e). These\nquantifications provide statistical validation and consistently demonstrate that\nRab14-CAMK2D axis is required for CTSD maturation.\nReviewer #3:\nThis article provides insights into how Rab14 contributes to pathogen clearance by\nregulating the CAMK2D-ATPase axis. Overall, the data are informative and support\nthe main conclusions. I have several comments that may help strengthen the manuscript.\nR: We thank the reviewer for the encouraging comments on our manuscript and\nfor the constructive suggestions to improve this manuscript.\n1. Recent work has shown that RAB14, together with other proteins, regulates\nautophagy by modulating the HOPS complex. This suggests that RAB14 may influence\nautophagy independently of lysosomal acidification. In Extended Data Fig. 8, it appears\nthat the maturation of LC3 puncta during autophagy is entirely regulated by the Rab14-\nCAMK2D axis. The authors may need to further discuss this point in the discussion. It\nwould be also good to explain more by new data if it’s necessary.\nR: We thank the reviewer for raising this critical point. As noted by the reviewer,\na recent study in MDCK epithelial cells demonstrated that Rab14 together with\nRab2 promote autophagosome-lysosome fusion through recruiting the HOPS\ncomplex (PMID: 38953305), indicating that Rab14 modulates autophagy\nindependent of the lysosomal acidification pathway that we revealed in our study.\nIndeed, in our previous Extended Data Fig. 8 (revised Supplementary Fig. 12), we\nmonitored the dynamic autophagy flux using the mCherry-GFP-LC3 tandem\nreporter. In this system, immature autophagosomes appear as yellow puncta (due\nto the overlap of green and red fluorescence), whereas mature autolysosomes\ndisplay only red puncta, as the GFP signal is quenched by the acidic environment\n(PMID: 38390831). Consequently, the ratio of yellow to total LC3 puncta serves as\na direct readout of autophagy flux, which is primarily dependent on lysosomal\nacidification. Therefore, regardless of whether Rab14 regulates autophagosome-\nlysosome fusion, its interaction with CAMK2D to control lysosomal acidification\nis the key determinant of the yellow-to-total fluorescence ratio.\nMoreover, to investigate whether the Rab14-CAMK2D axis regulates the\nautophagosome-lysosome fusion process in our system, we examined the\ncolocalization of LC3 with LAMP1 in Rab14+/+, Rab14-/-, Rab14+/+Camk2d-/-, and\nRab14-/-Camk2d-/- macrophages under uninfected or infected conditions, and our\nresults showed that the Rab14-CAMK2D axis did not affect the fusion between\nautophagosomes and lysosomes (revised Supplementary Fig. 13; lines 375–384).\nThus, Rab14-CAMK2D-mediated phosphorylation of V0a1 regulates the\nlocalization of V0a1 on lysosomal membrane to maintain lysosomal acidification\nby V-ATPase without affecting the autophagosome-lysosome fusion. It should be\nnoticed that our macrophage-based results are different from the previous study\nreporting that Rab14 promotes autophagosome-lysosome fusion in MDCK\nepithelial cells (PMID: 38953305), indicating that the function of Rab14 is cell\ntype-dependent, which is further supported in other studies. For instance, Rab14\npromotes the trafficking of glucose transporter 4 (GLUT4) from early\nendosomes to the Golgi apparatus following endocytosis in adipocytes (PMID:\n23444368; PMID: 26936971), while facilitating the GLUT4 translocation from the\nGLUT4 storage vesicles (GSV) compartment to the plasma membrane in muscle\ncells (PMID: 18701652). We have expanded our discussion accordingly (lines 444–451).\n2. In Fig. 2, the conclusions rely mainly on imaging analysis. It would be valuable to\ninclude complementary approaches to support the findings. In addition, some of the\nrepresentative images do not adequately reflect the statistical analysis. Selecting clearer\nimages would strengthen the presentation. Furthermore, about 50 cells were analyzed\nper biological replicate. Plotting the ratios from each individual cell would make the\ndata more convincing.\nR: We thank the reviewer for these constructive suggestions. To further validate\nthe subcellular localization of V0a1 observed by imaging in the previous Fig. 2, we\nperformed complementary subcellular fractionation assays to assess V0a1 levels.\nSpecifically, to test the conclusion from the previous Fig. 2a–e that V0a1 localizes\nto lysosomes in Rab14⁺/⁺ cells but to the ER in Rab14⁻/⁻ cells, we isolated ER and\nlysosomal fractions and confirmed that V0a1 is predominantly enriched in\nlysosomes in Rab14⁺/⁺ cells, while it is largely retained in the ER in Rab14⁻/⁻ cells\n(revised Supplementary Fig. 5a; lines 175–179, 745–751). Furthermore, regarding\nthe finding in the previous Fig. 2i that pathogen infection increases V0a1\ntrafficking to lysosomes in Rab14⁺/⁺, but not Rab14⁻/⁻ cells, we purified lysosomes\nfrom infected cells and confirmed a significantly greater enrichment of V0a1 on\nlysosomes from Rab14⁺/⁺ cells compared to those from Rab14⁻/⁻ cells (revised\nSupplementary Fig. 6a; lines 189–191). Additionally, we optimized our confocal\nmicroscopy experiments and provided clearer representative images that better\nalign with the quantitative data (revised Fig. 2). Finally, for quantification analysis,\nwe have plotted the values from each of the approximately 50 individual cells\nanalyzed per replicate (revised Fig. 2).\n3. For Fig. 3e, f, the same concern applies: some of the representative images are not\noptimal and could be replaced with more suitable ones to better reflect the quantitative\nanalysis.\nR: We thank the reviewer for raising this concern. We repeated and optimized the\nconfocal microscopy experiments in the previous Fig. 3e, f, and provided clearer\nand more representative images that better reflect the quantitative analysis\n(revised Fig. 3e, f).\n4. In Fig. 5i, the authors conclude that “Rab14 promotes the trafficking of V0a1 to\nlysosomes in a manner dependent on CAMK2D during pathogen infections.” However,\nthe data indicate that Rab14 also promotes V0a1 trafficking to lysosomes under non-\ninfection conditions. The conclusion should therefore be revised accordingly.\nR: We thank the reviewer for this insightful comment. As noted by the reviewer,\nRab14 indeed promotes V0a1 trafficking to lysosomes under non-infection\nconditions, as cells require a baseline level of lysosomal acidification to complete\nthe cellular degradation and recycling system under steady state (PMID:\n37543564). We have accordingly revised the conclusion of Fig. 5i in the manuscript\n(lines 344–349).\n5. LysoTracker (Invitrogen, L7528) is not a direct probe for intracellular pH. To validate\nthe key findings, the authors are encouraged to repeat some key experiments using\nLysoSensor probes, which are specifically designed for pH measurement.\nR: We thank the reviewer for this valuable suggestion. As advised, we performed\nlive-cell imaging assays using the LysoSensor Yellow/Blue probe, which mainly\nproduces yellow fluorescence in acidic lysosomes (low pH), but blue fluorescence\nin less acidic lysosomes (high pH) (PMID: 11444806). The results demonstrated\nthat compared with Rab14-/- macrophages, Rab14+/+ macrophages exhibited\nstronger yellow fluorescence but weaker blue fluorescence (revised Supplementary\nFig. 3b; lines 118–124, 861–866), confirming the conclusion that Rab14 is required\nfor lysosomal acidification.\nMinor points:\n1. Line 94: The reference to “Supplementary Fig. 1” may need to be deleted.\nR: As suggested by the reviewer, the reference to “Supplementary Fig. 1” has been\ndeleted in the revised manuscript (line 97).\n2. Extended Data Fig. 2b: It would improve clarity if the long-term and short-term LC3\nimaging results were presented together.\nR: We thank the reviewer for this helpful suggestion. We repeated the experiments\nin the previous Extended Data Fig. 2b–e, and provided the LC3 immunoblotting\nresults with both long-term and short-term exposures (revised Supplementary Fig.\n4a–d), consistently demonstrating that Rab14 promotes autophagy flux during\nmultiple pathogen infections.\nOnce again, we greatly appreciate the reviewers for having helped us improve\nthis manuscript tremendously."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have provided experimental data in response to my initial comments. This is a substantial contribution to the\nfield.", "reviewer_2": "The authors have addressed my concerns satisfactorily. I have no other comments and support publication of the\nmanuscript."}, "author_response": ""}]

Decision letter