A molecular switch in NAC prevents mitochondrial protein mistargeting by SRP

Domain: biology
Published: 2026-03-27
Editorial outcome: major_revision

Abstract
The nascent polypeptide-associated complex (NAC) co-translationally screens all nascent proteins and regulates their access to the signal recognition particle (SRP) to ensure the fidelity of protein targeting to the endoplasmic reticulum (ER). However, the mechanism by which NAC prevents the mistargeting of nascent mitochondrial proteins remains unclear. Here, we identify a molecular switch in NAC that allows its central barrel domain to adopt a stabilized conformation on ribosomes exposing a mitochondrial targeting sequence (MTS). Mutations of the MTS on the nascent chain or in the NAC switch region increase NAC barrel dynamics and reduce its binding to the ribosome. This leads to an impaired ability of NAC to prevent mistargeting by SRP and causes ER stress in human cells. Our work reveals how NAC detects nascent mitochondrial proteins early in translation and prevents their promiscuous access to SRP, elucidating the structural basis that underlies this role and providing mechanistic insights into protein targeting fidelity with broader implications for cellular proteostasis.

Introduction
During protein synthesis, nascent polypeptides are co-translationally sorted to distinct cellular destinations, a process critical for maintaining protein homeostasis1,2. This process begins on the ribosome, where the nascent polypeptide-associated complex (NAC) binds and screens all emerging nascent chains to regulate their localization and processing3,4. The majority of mitochondrial proteins are nuclear-encoded and synthesized in the cytosol5–7 where they must be properly screened by NAC to ensure accurate targeting. As part of this screening, nascent chain access to targeting factors, such as the signal recognition particle (SRP) that delivers translating ribosomes to the endoplasmic reticulum (ER), must be tightly controlled by NAC to prevent the mistargeting of mitochondrial proteins8. Despite its critical role in nascent chain sorting, whether and how NAC detects nascent mitochondrial precursor proteins and distinguishes them from ER-targeted proteins remain unclear.

NAC is an essential and conserved ribosome-associated complex in eukaryotes9–11 that is expressed at equimolar levels to ribosomes12. It is composed of the NACα and NACβ subunits13, which dimerize to form a central β-barrel domain14,15. NACβ anchors the complex to the ribosome with nanomolar affinity16 using a basic N-terminal tail that interacts with both ribosomal proteins and ribosomal RNA (rRNA)17–19. Previously, AlphaFold models fitted into lower resolution cryo-EM densities (~6-8 Å) suggested that two amphipathic helices (βh2 and αh1) in the NAC barrel domain lock together to form a clasp coordinated by a series of hydrophobic residues19 which mediate binding of the barrel domain near the polypeptide exit tunnel17,18. This positions the barrel domain to potentially interact with nascent chains emerging from the exit tunnel20,21 and coordinate their co-translational processing22–28 or targeting19,29,30.

One of the best-established functions of NAC is to prevent the mistargeting of nascent proteins to the ER8 by regulating the interaction, conformation, and activity of SRP16,19,29–31. SRP contains an M-domain, which recognizes an N-terminal hydrophobic ER targeting signal on nascent proteins emerging from the ribosome, and a GTPase NG-domain that dimerizes with the SRP receptor (SR) at the ER membrane16,29,32. Engagement of the M-domain with the signal sequence activates SRP for rapid assembly with SR to turn on ER targeting33,34 and prevents ER proteins from being mistargeted to mitochondria35. However, the specificity of ribosome binding and activation of SRP and SR is limited13,16. NAC uses dual mechanisms to regulate ER targeting. A flexibly tethered C-terminal UBA domain in NACα recruits SRP to translating ribosomes19, whereas the NAC barrel domain prevents the promiscuous activation of SRP by blocking the access of the SRP M-domain to the ribosome exit tunnel8,29,30. When an ER signal sequence emerges from the ribosome, the NAC barrel becomes destabilized, allowing SRP to displace it from the ribosome tunnel exit so that the nascent chain can be accurately targeted to the ER19. Unlike ER proteins, mitochondrial proteins do not destabilize NAC binding to the ribosome19. Thus, the specificity of nascent chain sorting by NAC is crucial to prevent mitochondrial protein mistargeting to the ER8.

A significant subset of mitochondrial proteins contain an N-terminal mitochondrial targeting sequence (MTS) that allows for their proper subcellular localization36,37. An MTS is a bipartite sequence motif characterized by clusters of positively charged residues on the polar face of a short amphipathic helix followed by a disordered sequence of 20-90 amino acids. The MTS, in particular the N-terminal basic amphiphilic helix, is recognized by receptors and the translocase at the outer mitochondrial membrane (TOM) and is often necessary and sufficient to direct nascent mitochondrial proteins for targeting and import6,7,38–40. However, whether NAC directly senses the MTS during translation prior to targeting and how NAC prevents promiscuous interactions of the MTS with SRP on the ribosome remains poorly understood.

Here, we determine the cryo-EM structure of human NAC bound to ribosomes translating a mitochondrial nascent chain with an N-terminal MTS. The structure reveals that the NAC barrel domain shifts to adopt a stabilized conformation docked at the rRNA helix H59 (H59-docked state) near the polypeptide exit tunnel mediated by a distinct set of ribosomal contacts that are distinct from previous observations of NAC bound to ribosomes translating an ER signal sequence or a cytosolic protein19,22,23. We identify a molecular switch in NACβ that allows NAC to alternate between different conformations. Single-molecule studies and biochemical assays indicate that mutations of the MTS disrupt the anchoring of the NAC barrel domain at the ribosome exit site, leading to the promiscuous activation of SRP on ribosomes exposing a nascent mitochondrial protein. Finally, we show that mutations in the NAC switch region fail to rescue to ER stress phenotypes that are associated with protein mistargeting in human cells. Our work uncovers a previously unappreciated ability of NAC to sense an MTS early during translation and the molecular mechanism employed by structural elements within NAC to prevent the mistargeting of nascent mitochondrial proteins.

Results
The NAC barrel adopts a distinct conformation on ribosomes translating an MTS
Previous studies established that NAC mediates proper protein sorting by preventing SRP access to ribosomes that do not carry signal sequences8,19,29. To first confirm if NAC deletion leads to mitochondrial protein mislocalization in human cells, NACβ knockout (KO) HEK293T cells were generated using CRISPR-Cas9 (Supplementary Fig. 1A). Immunofluorescence staining was conducted to assess the localization of OXA1L and HSP60, which are targeted to the inner mitochondrial membrane and mitochondrial matrix, respectively. In wild-type (WT) cells, both HSP60 and OXA1L co-localized with the mitochondrial marker TOM20 (Supplementary Fig. 1B-C). In contrast, NACβ-KO cells showed mislocalization of both proteins, with fluorescence signals accumulating outside mitochondria, indicating impaired mitochondrial protein sorting in the absence of NACβ.

Next, we asked how NAC engages with ribosomes carrying an MTS. Model MTS-containing nascent chains derived from OXA1L and HSP60 were generated by stalling ribosomes during translation with the XBP1 arrest peptide41 or on mRNA lacking a stop codon42, respectively. NAC was added to salt-washed stalled ribosome-nascent chain complexes (RNCs), exposing the N-terminal MTS (RNC ) of OXA1L (74 residues) or HSP60 (62 residues), to obtain a homogeneous population suitable for structural analysis. As a control, an MTS deletion mutant of OXA1L was also purified (Supplementary Fig. 2). Single particle cryo-EM analysis was used to visualize the interactions between NAC and ribosomes translating mitochondrial nascent chains (Supplementary Table 1).

Initial 2D and 3D classifications yielded heterogenous 80S particles with different translation states. Further iterative 3D variability analyses were conducted using focused masks to sort for ribosomes translating mitochondrial nascent chains, which resulted in a final set of particles containing density for P-site tRNA, nascent chain, and NAC (Supplementary Fig. 3A-B, Supplementary Fig. 4A-B). The cryo-EM structures of both OXA1L- and HSP60-RNC MTS resolved NAC bound next to the polypeptide exit tunnel. The overall resolution of the two maps was ~2.6 Å and ~3.0 Å. NAC was resolved between 3-6 Å (Supplementary Fig. 3C-D, Supplementary Fig. 4C-D) in a similar conformation on the ribosome for both MTS constructs interacting with rRNA helix 59, termed the H59-docked state (Supplementary Fig. 5A-C). Since the NAC barrel domain was better resolved in the OXA1L-RNC map, this map was used for further interpretation of the structure.

The NAC barrel was docked near the polypeptide exit tunnel (red asterisk, Fig. 1A), consistent with its role in protecting and scanning emerging nascent chains8,21. Cryo-EM density corresponding to the nascent chain was present in the polypeptide exit tunnel, but the nascent chain including the MTS was not resolved outside the ribosome due to flexibility (Fig. 1A, Supplementary Fig. 6A-B). In the current H59-docked state, NAC was stabilized by a series of contacts between NACβ and the ribosome (Fig. 1B -E) that were not previously observed in the structure of NAC bound to ribosomes translating an ER signal sequence (RNC )19.

NACβ contains a conserved N-terminal RRKKK motif, which is essential for high-affinity ribosome binding of the complex17. This ribosome ‘anchor’ of NAC was previously observed to intercalate around the C-terminal tail of eL22, forming extensive interactions between the ribosomal RNA (rRNA) and the ribosomal protein eL1919. In addition to the previously observed ribosome contacts, we resolved an additional insertion of the first 4 N-terminal residues of NACβ wedged between rRNA helix 59 (H59) and eL22, which forms the fluke of the NAC anchor (Fig. 1C, Supplementary Fig. 7A-B). A combination of polar and stacking interactions mediate the contacts with eL22, consistent with crosslinking data21.

These previously unresolved contacts are established following a large conformational shift of the NAC barrel towards H59 (colored grey vs. white, Fig. 1F). In particular, the base of the barrel that directly contacts the ribosome moves by 10 Å away from the polypeptide exit tunnel. To accommodate this shift, H59 undergoes a conformational change where it lifts away from the ribosome by 9 Å (red arrows, Fig. 1F). In this state, the rRNA bases G2706 and U2707 are flipped out following rearrangement of the rRNA (red arrows, Fig. 1G) allowing NAC to dock at rRNA H59.

Two predicted amphipathic helices in the NAC barrel domain (βh2 and αh1) and part of the linker on NACβ that connects the barrel domain to the anchor (Fig. 1B) were resolved at side-chain resolution and built de novo (Fig. 1D and Supplementary Fig. 8A-C). These amphipathic helices form a clasp containing a hydrophobic core (Supplementary Fig. 8A) and mediate the interactions (described below) that stabilize the NAC barrel on the ribosome. The H59-docked state is distinct from previous structures of NAC bound to ribosomes translating and displaying an ER signal sequence (H59-undocked state), in which NAC is further away from H59 and both the hydrophobic clasp and contacts with the ribosome were destabilized by the emergence of a signal sequence and thus were not well resolved19.

A molecular switch regulates NAC binding to the ribosome
Several residues in NAC βh2 and the preceding linker, conserved among higher eukaryotic organisms, were identified as mediating interactions with the ribosome (Fig. 2A). In the NACβ linker region, His34 inserts below H59 to lock NAC in a stable conformation on the ribosome (Fig. 2A), while the side chain of Arg35 interacts with the flexible C-tail of eL22 (Fig. 2B and Supplementary Fig. 8A). Furthermore, Lys42 is within range for electrostatic interactions with the phosphate backbone of rRNA H59, while Phe46 stacks with Asn34 on eL19 (Fig. 1E, Supplementary Fig. 8C). These interactions are formed in the H59-docked state of the RNC MTS structure following a conformational change in the NAC βh2 and the preceding linker, when compared to the previous RNC structure (Fig. 2B).

To investigate the role of these previously uncharacterized contacts in ribosome binding, residues mediating NAC barrel domain interactions with the ribosome (His34, Arg35, Lys42, and Phe46) were mutated to either Glu/E or Ala/A and purified (Supplementary Fig. 9A-B). We tested the ability of the NAC mutants to interact with human ribosomes isolated from HEK293 cells using an in vitro co-sedimentation assay. Both single and double mutations in the NACβ linker (His34 and Arg35) and the amphipathic helix βh2 (Lys42 and Phe46) significantly reduced NAC association with ribosomes when compared to wild-type (WT) NAC (Fig. 2C-D, Supplementary Fig. 9C-D).

Together, our structural and biochemical data reveal a molecular switch within NACβ that undergoes a conformational change involving the linker residues (His34 and Arg35) and the amphipathic helix βh2. Upon binding ribosomes displaying an MTS, this switch engages with rRNA helix H59 and stabilizes the positioning of the NAC barrel near the polypeptide exit tunnel.

The molecular switch alters NAC barrel dynamics on the ribosome
We next directly assessed how contacts of the switch region (Fig. 3A) influence NAC conformations on the ribosome by conducting single-molecule total internal reflection fluorescence microscopy (smTIRFM) studies using OXA1L RNC immobilized on a glass coverslip surface (Fig. 3B). This approach selectively monitors populations of WT or mutant NAC that are bound to surface-immobilized ribosomes and excludes unbound NAC.

The position of the NAC barrel at the ribosome tunnel exit was measured by Förster resonance energy transfer (FRET) between a donor dye (Cy3B) on the NAC barrel domain and an acceptor dye (Atto647N) incorporated at residue 39 in the OXA1L nascent chain. This residue is 35 amino acids from the C-terminus of the nascent chain and places the dye at the polypeptide exit tunnel (Fig. 3B). Hidden Markov modeling (HMM) of the fluorescence time traces showed dynamic transitions of the NAC barrel between three states, with low, medium, and high FRET efficiency, on the second timescale (Fig. 3C). The majority of WT NAC displayed long-lived high FRET traces with brief transitions to medium and low FRET states (Fig. 3C), indicating stable docking of the barrel in close proximity to the tunnel exit. In contrast, NAC bearing mutations in the NACβ linker (HR:AA) or βh2 (KF:AA) more frequently sampled the medium and low FRET states (Fig. 3C). These differences are reflected in the FRET histograms, which show that WT NAC resides primarily in the high FRET state, whereas the FRET distribution for NAC switch mutants shifted to medium- and low-FRET states (Fig. 3D-E). These results demonstrate that the docking of the NAC barrel at the ribosome tunnel exit was impaired by mutations in the switch region.

We further measured the kinetic stability of NAC interactions with RNC using smTIRFM. Over 80% of WT NAC dissociated from RNC with a rate constant of ~0.14 s-1, while the remainder dissociated at a faster rate (Supplementary Fig. 10A). The more-stably bound population (slow-dissociating) decreased to ~40% and ~20%, respectively, with NAC mutants HR:AA and KF:AA (Supplementary Fig. 10B). Thus, mutations in the NAC switch region impaired proper barrel docking on the ribosome surface, and the faster dissociation of these mutants compared to WT NAC are consistent with co-sedimentation assays showing reduced mutant binding to ribosomes (Fig. 2C-D). We note that despite the weakened binding, the association of HR:AA and KF:AA were still confidently detected under smTIRFM with < 5 nM NAC present, indicating that both NAC mutants still bind ribosomes with high affinity. Taken together, these data suggest that the molecular switch controls the conformational dynamics of the NAC barrel on the ribosome.

MTS mutations destabilize the NAC barrel on the ribosome
We next asked whether the stabilized conformation of the NAC barrel domain in the H59-docked state at the ribosome exit site is influenced by the presence of an MTS. To this end, we either deleted the amphiphilic helical segment of the MTS in the OXA1L nascent chain (OXA1L ) or replaced this region with the ER signal sequence from a bona fide SRP substrate preprolactin (OXA1L ) (Fig. 4A). smTIRFM experiments were performed to monitor the NAC barrel conformation on RNCs carrying the mutated MTS. Both mutations led to increased dynamics of the NAC barrel, substantially increasing the population in low and medium FRET states compared to that on RNC bearing the WT OXA1L MTS (Fig. 4B-C). In addition, the more stably bound (slow-dissociating) NAC population on these RNCs decreased to ~40% and ~60% for the OXA1L ∆MTS and OXA1L MTS-to-ISS nascent chain, respectively, compared to 80% on OXA1L WT (Fig. 4D-E).

Based on the smFRET data, we hypothesized that mutation of the MTS destabilizes the NAC barrel, similar to previous structures of NAC bound to ribosomes displaying the nascent chain of an SRP substrate, preprolactin19. We therefore determined the structure of NAC bound to ribosomes translating OXA1L (Supplementary Fig. 11A-D). A 3D variability analysis of the OXA1L dataset similar to that performed for the MTS dataset revealed multiple NAC classes exhibiting barrel movements on the ribosome (Fig. 4F, Supplementary Fig. 12A-B). In addition to the H59-docked state, we observed the H59-undocked state similar to a previous structure of NAC on ribosomes translating an ER substrate19 where the barrel moves away from H59 and towards the polypeptide exit tunnel (Supplementary Fig. 13A-C). This state showed less-defined EM-densities for the two amphipathic helices of NAC implying increased dynamics in this region, consistent with previous observations19. Collectively, these results indicate that the H59-docked state is promoted by the presence of an MTS which is likely crucial for preventing improper targeting of MTS-containing nascent chains by SRP.

NAC mutants fail to regulate SRP conformation on RNCs
We next tested whether the contacts made by the NAC switch region are important for preventing the mistargeting of ribosomes exposing a nascent MTS. SRP is activated for ER targeting when the SRP54 M-domain engages the ER signal sequence emerging from the exit tunnel, which induces SRP to adopt a ‘Proximal’ conformation in which the SRP54 NG-domain docks at uL23 and optimally associates with the SRP receptor at the ER16,34. Previously, we showed that NAC blocks SRP from adopting the targeting-active proximal conformation on ribosomes lacking an ER targeting signal and thus prevents mistargeting16. In the current conformation of NAC on RNC , the NAC barrel is stabilized at the exit tunnel and would effectively block the binding of SRP54 M-domain at the exit tunnel (Fig. 5A), thus preventing the activation of SRP16,32. We hypothesize that the loss of the molecular switch would impair this sorting function of NAC, by reducing the stable docking of the NAC barrel at the polypeptide exit tunnel.

We conducted smTIRFM experiments to observe the conformational dynamics of SRP bound to ribosomes translating either an ER or a mitochondrial nascent chain. The proximal conformation of SRP was detected using FRET between a donor dye (Atto550) and an acceptor dye (Atto647N) labeled on SRP19 and SRP54, respectively (Fig. 5B)16,43. On ribosome bearing an ER signal sequence from preprolactin, SRP is dominated by a high FRET state that reports on the proximal conformation, both in the absence and presence of NAC (Fig. 5C), consistent with previous results16. On ribosomes exposing the MTS of OXA1L, SRP is distributed between three conformations, with ~40% of SRP in the high-FRET, proximal conformation that is targeting-active (Fig. 5D). The proximal conformation of SRP on RNC was abolished by WT NAC, whereas both NAC mutants failed to regulate the conformation of SRP (Fig. 5D-E). Thus, mutations in the switch region abolished the ability of NAC to prevent the mis-activation of SRP on ribosomes translating an MTS.

Previous work demonstrated that NAC knockdown led to the mistargeting of mitochondrial proteins to the ER, which induced modest ER stress and elevated expression of ER chaperones BiP and PDI8. We therefore tested the importance of the NAC molecular switch in preventing protein mistargeting to the ER by assessing the levels of ER chaperones and the splicing of the XBP1 mRNA, both of which report on ER stress44,45. We first showed that NACβ-KO HEK293T cells displayed increased levels of the ER chaperones, BiP and PDI (Supplementary Fig. 14A), as well as XBP1 mRNA splicing (Supplementary Fig. 14B). We then reintroduced either WT or mutant NACβ in the NACβ-KO cells. Overexpression of WT NACβ partially restored the ER stress markers, while both the NACβ HR:AA and KF:AA mutants failed to rescue the ER stress phenotype (Fig. 5F-G).

To assess whether ER stress in NACβ-KO cells arises from mistargeting of mitochondrial proteins to the ER where they become aberrantly glycosylated, we performed concanavalin A (ConA) pulldowns of glycosylated proteins from whole-cell lysates8. We observed a substantial increase in glycosylated HSP60 accumulation in NACβ-KO HEK293T cells relative to WT cells (Supplementary Fig. 15A-B). Overexpression of WT NACβ partially rescued glycosylated HSP60 accumulation (0.3 for WT versus 0.5 for rescue compared to KO levels), whereas the NACβ switch mutants failed to rescue the phenotype to the same levels (1.4 for the NACβ HR mutant and 0.8 for the KF mutant). Taken together, our results indicate that the NAC molecular switch prevents non-specific binding and activation of SRP to MTS-containing RNCs, and when perturbed, can lead to mitochondrial protein mistargeting which induces modest ER stress in human cells.

Methods
Immunofluorescence
HEK293T cells were cultured on Millicell EZ SLIDE 8-well chamber (Millipore # PEZGS0816) for 24 hours. The cells were fixed by 4% paraformaldehyde (PFA) for 10 minutes at room temperature, followed by three washes with PBS. The fixed cells were simultaneously incubated with primary antibodies in blocking buffer (0.3% donkey serum and 0.25% Triton X-100 in PBS) overnight at 4 °C. Following three washes with PBS, the samples were incubated with secondary antibodies for 2 hours at room temperature. Mounting was performed with mounting medium containing DAPI (Vector Laboratories, cat no. H-1200) and Fisherfinest Premium Cover Glasses (Fisher Scientific, cat no. 12-548-5P). Images were captured using the Zeiss LSM 980 confocal microscope at the University of Virginia Advanced Microscopy Facility. Antibodies for immunostaining were as follows: anti-HSP60 (mouse, Invitrogen, cat no. MA3-012, 1:200), anti-TOM20 (rabbit, Proteintech, cat no. 11802-1, 1:200), anti-OXA1L (rabbit, Proteintech, cat no. 21055-1, 1:200), anti-TOM20 (mouse, Abcam, cat no. ab56783, 1:200). Secondary antibodies for fluorescent immunostaining (all 1:500) were as follows: Anti-rabbit IgG Alexa Fluor 488 (Jackson ImmunoResearch, cat no. 711-545-152), anti-Mouse IgG Alexa Fluor 555 (Invitrogen, cat no. A32773).

NAC Purification
A construct encoding for 6xHis-NACα and NACβ in a pET28b vector22 was expressed in E. coli BL21-CodonPlus DE3 competent cells (Agilent, cat no. 230245). QuickChange (Agilent, cat no. 210518) site-directed point mutagenesis was conducted to generate mutations in NACβ. The following procedures were used to purify all NAC variants. Cells were grown to OD600 0.6-0.9 at 37 °C and induced with 1 mM IPTG at 18 °C overnight. Cells were pelleted, resuspended in 15 mL of lysis buffer (50 mM HEPES-KOH pH 7.5, 1M NaCl, 10% Glycerol, 6 mM β-mercaptoethanol, protease inhibitor (Roche, complete mini, cat no.11836153001)), and lysed using a French press. The lysate was centrifuged using a TI 50.2 rotor at 20,000 rpm for 30 min at 4°C (2X). The supernatant was loaded on a 5 mL HisTrap HP column (Cytiva, cat no. 17524802) using a P1 pump at 4°C and washed with 1-2 column volumes (CV) of Nickel A buffer (50 mM HEPES-KOH pH 7.5, 1M NaCl, 45 mM Imidazole, 10% glycerol, 6 mM β-mercaptoethanol). The column was transferred to an Akta Pure fast protein liquid chromatography system (Cytiva) and washed for an additional 5 CV in nickel A buffer. Purified proteins bound to the column were eluted in a step gradient using 2-3CV of 15% and 30% nickel B buffer (50 mM HEPES-KOH pH 7.5, 150 mM KOAc, 300 mM Imidazole, 10% glycerol, 6 mM β-mercaptoethanol).

Fractions from the 30% peak were pooled and added to 7K MWCO dialysis tubing (ThermoFisher, cat no. 68700) with 3C protease (Genescript, cat no. Z03092) to cleave the N-terminal His tag on NACα. Pooled fractions were dialyzed overnight at 4°C in dialysis buffer (50 mM HEPES-KOH pH 7.5, 150 mM KOAc, 10% glycerol, 6 mM β-mercaptoethanol). Dialyzed proteins were loaded onto a HiTrap Q HP column (Cytiva, cat no. 17115401) using a P1 pump and the column was washed with 1-2 CV of ion exchange buffer A (50 mM HEPES-KOH pH 7.5, 2 mM DTT, 2 mM EDTA, 10% glycerol). The column was moved to the Akta and bound proteins were eluted from the column using a linear gradient of ion exchange buffer B to 50% (50 mM HEPES-KOH pH 7.5, 1 M NaCl, 2 mM DTT, 2 mM EDTA, 10% glycerol). Fractions eluted with ~30% ion exchange buffer B were combined and concentrated using an Amicon Ultra 4 mL centrifugal filter (Millipore, cat no. UFC801024). One volume of ion exchange buffer A was added to the sample and the concentration of NAC was determined on the Nanodrop (ThermoFisher) using a molar extinction coefficient, 2980 M-1 cm-1. The samples were snap frozen in liquid nitrogen and stored at -80 °C. Purified NAC was resolved by SDS-PAGE and gels were stained with Coomassie R-250 (Sigma, cat no. B-0149).

NAC labeling
WT NACα/NACβ(S57C) and ribosome-binding mutants were labeled with maleimide-Cy3B (Cytiva). The protein was exchanged into Labeling buffer (50 mM KHEPES, pH 7.5, 100 mM NaCl, 1 mM TCEP, 20% glycerol) and incubated with a 5-fold molar excess of dye for 2 hours at room temperature. Free dye was removed using a G-25 Sephadex size exclusion column (GE Healthcare). Fractions containing labeled NAC were identified by SDS-PAGE, pooled, and concentrated to 100-250 μM.

Purification and labeling of SRP subunits
Purification of SRP9/14, SRP19, SRP54, and SRP68/72 were described previously43. The single cysteine mutants SRP19(K64C) and SRP54(S12C) were purified as for WT proteins and labeled with maleimide-Atto550 and maleimide-Atto647N (ATTO-TEC), respectively, following published protocols16,43. Briefly, SRP19(K64C) and SRP54(S12C) were incubated with 2 mM DTT for 30 minutes at 25 °C. Reduced proteins were exchanged into the Labeling buffer by passing twice through a micro Bio-Spin column packed with Bio-Gel P-6 resin (Bio-Rad). Proteins were incubated with an 8-fold molar excess of maleimide dye for 3 hours at 4°C. The labeling reaction was quenched with 2 mM DTT, and excess dye was removed using the G-25 Sephadex Fine size exclusion column (GE Healthcare) run in the labeling buffer (50 mM KHEPES, pH 7.5, 300 mM NaCl, 2 mM EDTA, 1 mM TCEP, 10% Glycerol). Fractions containing labeled SRP subunits were identified by SDS-PAGE, pooled, and concentrated to ~40 μM.

SRP assembly
SRP was assembled following the protocol established by Lee et al43. Purified and folded 7SL SRP RNA was incubated in the binding buffer (20 mM Tris, pH 7.5, 300 mM KOAc, 5 mM Mg(OAc)2, 5 mM DTT, 10% Glycerol) at 37 °C. Subsequently, the assembly reaction was performed in the HKMN buffer (50 mM KHEPES, pH 7.5, 500 mM KOAc, 5 mM Mg(OAc)2, 1 mM DTT, 0.02% NIKKOL) with the sequential addition of the protein subunits in the following order: SRP19(K64C)-Atto550 for 10 minutes, SRP68/72 together with SRP9/14 for 10 min, and SRP54(S12C)-Atto647N for 30 minutes at 37 °C. Holo-SRP was purified on a DEAE-Sephadex anion exchange column (Sigma). Fractions containing fully assembled SRP were eluted in a buffer containing 600 mM KOAc, identified by A260 measurements, pooled, and stored at -80 °C. The activity of purified SRP was tested using a translocation assay with preprolactin as the SRP substrate using salt-washed ER microsomes incubated in wheat germ extracts16.

PCR and in vitro Transcription
A construct encoding for the mitochondrial matrix protein HSP6021, and gblocks (Integrated DNA Technologies) encoding the N-terminus of mitochondrial inner membrane protein OXA1L (AAs 2-51, AMGLMCGRRELLRLLQSGRRVHSVAGPSQWLGKPLTTRLLFPAAPCCCRP) or OXA1L (deletion of MTS residues LMCGRRELLRLLQSGRRV), was PCR amplified to include the upstream T7 promoter, 3X-FLAG tag, and the N-terminus of each nascent chain. OXA1L constructs used for cryo-EM contained a C-terminal modified XBP1 ribosome stalling peptide (AAs 237-260, DPVPYQPPFLCQWGRHQCAWKPLM). PCR products were then cleaned up using the QIAquick PCR Purification Kit (Qiagen, cat no. 28104). The resulting purified PCR products were in vitro transcribed for 4 hours at 38 °C, using 1.1 mg/mL T7 polymerase in reaction buffer (40 mM Tris-HCl pH 7.6, 5 mM ribonucleoside triphosphates, 6 mM MgCl2, 2 mM spermidine, 1 mM DTT, 0.04 U/µL RNase Inhibitor) or with the T7 HiScribe kit (NEB, cat no. E2040S). Following the reaction period, the precipitate was pelleted at ~22,000 x g for 5 minutes at 4°C. The supernatant was transferred to a sterile 1.5 mL tube and 6M LiCl was added 1:1 for 1 hour on ice. After the incubation period, the mixture was centrifuged at ~22,000 x g for 20 minutes at 4°C and the supernatant was discarded. The pellet was washed with 200 µL of ice cold 70% ethanol and centrifuged at ~22,000 x g for 5 minutes at 4°C. The supernatant was discarded and 100 µL of sterile ultrapure water was used to resuspend the pellet. The sample was placed on ice and 40 µL of 2.8 M NaOAc and 300 µL 95% ice cold ethanol were added. The mixture was incubated on ice for 5 minutes and centrifuged at ~22,000 x g for 30 minutes at 4°C. The resulting pellet was washed with 200 µL of 70% ethanol and centrifuged at ~22,000 x g for 5 min at 4°C. The supernatant was discarded, and the resulting purified RNA was resuspended in 100 µL of sterile ultrapure water.

RNC purification for cryo-EM
Rabbit reticulocyte lysates (Promega, cat no. L4540) were reacted with HSP60 or OXA1L RNA after diluting the lysate in buffer (final concentration: RNase Inhibitor (Promega) 0.04 U/µL, 0.5X protease inhibitor (Promega), 24 µM amino acid mix, 81 mM KCl, 0.67 mM Mg(OAc)2). The reaction was incubated at 32 °C for 25 minutes and then placed on ice.

A 1 mL polypropylene gravity column (Qiagen, cat no. 34924) was loaded with Anti-DYKDDDK G1 affinity resin (GenScript, cat no. L00432 or Sigma, cat no. A2220). The column was rinsed with 10 bead volumes of 1X PBS followed by 10 bead volumes of low-salt wash buffer (50 mM HEPES-KOH pH 7.7, 100 mM KCl, 10 mM MgCl2). The RRL reaction was added to the column and incubated for 2 hours at 4°C with rotation. After the incubation period, the cap and side walls of the gravity column were rinsed with high-salt wash buffer (50 mM HEPES-KOH pH 7.7, 750 mM KOAc, 10 mM Mg(OAc)2, 0.1% Triton-X, 1 mM DTT). The reaction supernatant was run through the column and the beads were subsequently washed with 10 bead volumes of high-salt wash buffer (2X) followed by 10 bead volumes of low-salt wash buffer (2X). RNCs were incubated with elution buffer (50 mM HEPES-KOH pH 7.7, 100 mM KOAc, 15 mM Mg(OAc)2) containing 0.25 mg/mL FLAG peptide for 15 minutes at RT (5X). The eluted fractions were placed on ice and combined to a total volume of 500 µL and centrifuged at 100,000 rpm for 1 hr at 4°C using the TLA120.1 rotor (Beckman). The pellet was resuspended in base elution buffer by gently pipetting up and down. The ribosome concentrations were determined using RNA reading at A260 on the Nanodrop. RNCs were snap frozen in liquid nitrogen and stored at -80 °C.

RNC labelling and purification for smTIRFM
In vitro translations used a pUC19 vector containing, from 5’ to 3’, a T7 promoter, encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), 3xFLAG-tag, and 3C protease site. DNA fragment encoding the nascent chain of preprolactin or OXA1L was cloned after the 3C protease site using Gibson assembly. In the OXA1L construct, an amphipathic helical region (MTS) of the OXA1L presequence (LMCGRRELLRLLQSGRRV, residues 4-21) was deleted, whereas in the OXA1L the deleted region was swapped with signal sequence of preprolactin (LLLLLLVSNLLL, residues 13-24). For fluorescent labeling of the RNCs, an amber codon was introduced at residue 39 in the OXA1L nascent chain using QuickChange mutagenesis.

PCR fragments encoding the region from the T7 promoter to Ser74 of OXA1L or Phe80 of preprolactin were transcribed using the T7 MegaScript protocol in the presence of 5 mM 5’-biotin-G-monophosphate (TriLink). The resulting mRNAs were translated in rabbit reticulocyte lysate (RRL, Green Hectares) supplemented with 1 µM Methanosarcina mazei pyrrolysine synthase (MmPylRS), 10 mg/L M. mazei amber suppressor tRNA (MmPyltRNA), and 100 µM axial trans-cyclooct-4-en-L-lysine (TCOK, SiChem), for 30 minutes at 32 °C16.

The translation reaction was layered on a High Salt Sucrose Cushion (50 mM KHEPES, pH 7.5, 1 M KOAc, 15 mM Mg(OAc)2, 0.5 M Sucrose, 0.1% Triton, 2 mM DTT) at a volumetric ratio of 2:3. The ribosomal fraction was pelleted by ultracentrifugation (100k rpm for 45 minutes at 4°C) in a TLA100.3 rotor (Beckman Coulter). Pellets were resuspended in RNC buffer (50 mM KHEPES, pH 7.5, 150 mM KOAc, 2 mM Mg(OAc)2) to 1 µM ribosome concentration and incubated with 1 µM tetrazine-conjugated Atto647N dye (Jena Biosciences) for 30 minutes at 25 °C, to allow for the Diels-Alder cycloaddition reaction. Labeled RNCs were incubated with anti-DYKDDDK magnetic agarose (Pierce) pre-equilibrated in RNC buffer for 1 hour at 4°C with constant rotation. Beads were washed sequentially with 10 bead volumes of RNC buffer containing 300 mM KOAc, RNC buffer containing 0.1% Triton, and RNC buffer. RNC was eluted with 1.5 mg/ml 3xFLAG (DYKDDDK) peptide for 30 minutes at 4°C with constant rotation. The eluted RNCs were incubated with 1 µM 3C protease (GoldBio) for at least 2 hours at 25°C, and sedimented using a High Salt Sucrose Cushion at a volumetric ratio of 2:3 at 100k rpm for 30 minutes at 4°C in a TLA120.2 rotor (Beckman Coulter). Pelleted RNC was resuspended in storage buffer (50 mM KHEPES, pH 7.5, 150 mM KOAc, 5 mM Mg(OAc)2, 0.04% NIKKOL, 2 mM DTT) to ~100 nM (ribosome concentration). The presence of fluorescently labeled nascent chain was confirmed by SDS-PAGE and fluorescence scanning for Atto647N signal (Typhoon biomolecular imager, Cytiva). Fluorescently labeled RNCs were snap frozen in liquid nitrogen and stored at -80 °C.

Single-molecule TIRF microscopy
Surfaces of pure quartz imaging slides and coverslips were aminosilanized with vectabond (Vector lab) and coated with biotin-PEG (Laysan Bio). Slides were passivated with passivation buffer (1X Tween-20, 1 mg/ml BSA) for at least 1 hour at 25 °C, washed with 50 mM KHEPES, pH 7.5, and incubated with 0.5 mg/ml NeutrAvidin (ThermoFisher) for 10 minutes. Unbound NeutrAvidin was washed away with Imaging Buffer, which constitutes Assay Buffer (50 mM KHEPES, pH 7.5, 150 mM KOAc, 5 mM Mg(OAc)2, 0.04% NIKKOL, 2 mM DTT) supplemented with 1 mg/ml BSA, 4 mM Trolox, 2.5 mM protocatechuic acid (PCA), and 50 nM protocatechuate-3,4-dioxygenase from Pseudomonas sp. (PCD, Sigma)48. For assays involving SRP, the imaging buffer further included 200 μM non-hydrolyzable GTP analog, guanosine-5′-[(β,γ)-imido]triphosphate (GppNHp).

RNC with 3’-biotinylated mRNA was diluted to 1.5 nM in Imaging Buffer and immobilized on NeutrAvidin-coupled slides for 10 minutes at 25 °C. For experiments with NAC, RNC-coated slides were then flushed with 2 nM WT or mutant Cy3B-labeled NAC in Imaging Buffer. For experiments involving NAC and SRP, 1.5 nM doubly labeled SRP and 2 nM unlabeled WT or mutant NAC were incubated in Imaging Buffer for 5 minutes and loaded onto slides with immobilized RNCs.

Movies were recorded using MicroManager on a custom-built TIRF microscopy setup49. The presence of immobilized Atto647N-conjugated RNCs was confirmed by excitation at 635 nm. Movies were recorded by excitation at 532 nm (donor dye; Atto550 or Cy3B) in single-excitation mode and detection of both the donor and acceptor channels, with a temporal resolution of 50 ms.

Analysis of smTIRF microscopy data
Donor and acceptor channel image series were aligned and analyzed using iSMS software50. FRET efficiencies were calculated based on the raw intensity of donor and acceptor fluorescence time traces and were corrected for background noise and 𝛾 factor, which accounts for the difference in quantum yield between the dyes and leakage of donor fluorescence to the acceptor channel. Apparent FRET efficiency (Eapp) was calculated using Equation 1, Eapp = IDD / (IDD + IAD) in which IDD and IAD are the fluorescence intensities of the donor and acceptor dye, respectively, upon excitation of the donor. FRET traces from all movies of the same sample were combined and analyzed using Hidden Markov Modeling (HMM) available in the iSMS software. The number of FRET states was established by applying Bayesian information criterion. These analyses yielded three FRET populations and the mean FRET efficiency for each state. These parameters were used for Gaussian fitting of the FRET histograms according to Eq 2, PDF = ∑ni=1 Ai ∙ 1 / (σi√2π) ∙ e -1/2 ((E-μi)/σi)2 in which PDF is the probability density function, n is the number of FRET states detected and is set to 3 (determined by HMM), Ai is the weight of the ith Gaussian, and σi and μi are the standard deviation and center (determined by HMM) of the ith Gaussian.

To determine the residence time of NAC on RNC, cumulative fluorescence intensity signal (Icumulative) was calculated using Eq 3, Icumulative = IDD + 𝛾 ∙ IAD and plotted over time for each trace. Colocalization of labeled NAC and RNC identified by Icumulative over background noise. Two-state HMM was then used to distinguish bound versus dissociated NAC and determine the dwell time of each colocalization event (t). The cumulative probability distribution of t was fitted to Eq 4, f(t) = Afast ∙ e-kfast ∙ t + Aslow ∙ e-kslow ∙ t in which Afast and Aslow are the amplitudes of the fast and slow-dissociating populations, and kfast and kslow are the respective dissociation rate constants.

Cryo-EM sample preparation and data acquisition
FLAG-SUMO-HSP60 RNCs (~350 ng/uL) were reacted with 260 nM SUMO protease (Trialtus, cat no 30-1030) at 30°C for 20 minutes. NAC was added at a final concentration of 2 µM in reaction buffer (50 mM HEPES-KOH pH 7.7, 100 mM KOAc, 15 mM Mg(OAc)2, 0.01875% GDN) for 20 minutes at 30°C. The reaction was moved to ice prior to freezing grids. Quantifoil R2/1 300 mesh copper grids (Quantifoil, cat no. Q350CR1) were freshly coated with 3.3 nm carbon and glow discharged at 15 mA for 15 seconds using an EMS glow discharger. A 5 µL drop of the NAC-RNC reaction was incubated for 1 minute on the grid at 100% relative humidity and 4°C before blotting. The grid was subsequently plunge frozen into a cup filled with liquid ethane pre-cooled to liquid nitrogen temperatures. Cryo-EM data was collected on the ThermoFisher Glacios electron microscope operated at 200 kV and equipped with a Falcon4 direct electron detector at a total magnification of 120,000x using a defocus range of -2.2 to -1.2 and a step size of 0.2. Movies were collected at a pixel size of 1.2 Å with a dose of 50 e/Å2 across 40 frames per movie.

FLAG-3C-OXA1L and FLAG-3C-OXA1L RNCs (~200-250 ng/uL) were reacted with 250 nM 3C protease at 30°C for 20 minutes. NAC was added at a final concentration of 1 µM in reaction buffer (50 mM HEPES-KOH pH 7.7, 100 mM KOAc, 15 mM Mg(OAc)2, 0.02% C12E8) for 20 minutes at 30°C. The reaction was moved to ice prior to freezing grids. Quantifoil R2/1 300 mesh copper grids (Quantifoil, cat no. Q350CR1) were freshly coated with 3.3 nm carbon and glow discharged at 15 mA for 15 seconds using an EMS glow discharger. A 5 µL drop of the NAC-RNC reaction was incubated for 1 minute on the grid at 100% relative humidity and 4°C before blotting. The grid was subsequently plunge frozen into a cup filled with liquid ethane pre-cooled to liquid nitrogen temperatures. Cryo-EM data was collected on the ThermoFisher Krios electron microscope operated at 300 kV and equipped with a K3 direct electron detector at a total magnification of 105,000x with an energy filter slit width of 10 eV, using a defocus range of -1.4 to -0.8 with a step size of 0.2. Movies were collected at a pixel size of 0.83 Å with a dose of 32 e/Å2 (OXA1L ) or 40 e/Å2 (OXA1L ) across 40 frames per movie.

Cryo-EM data processing strategy for HSP60 RNC
Movies were motion-corrected, dose-weighted, and the contrast transfer function (CTF) was estimated in CryoSPARC51,52. Template picker was used to pick particles with a diameter of 250 Å from the resulting micrographs. A total of 1,366,067 particles were extracted from 8,780 micrographs at a box size of 512 x 512 pixels. The extracted particles underwent 2D classification, with ribosome classes selected for further processing. A homogenous refinement of the selected 519,343 particles yielded a consensus 80S ribosome map at 2.97 Å. Following refinement, translating ribosomes with P-site tRNA density were classified using a spherical mask on the A/P/E by conducting a 3D variability analysis in CryoSPARC53. Particles with a stable P-site tRNA density were refined separately and subjected to an additional 3D variability analysis, using a spherical mask on the exit tunnel to identify particles with strong NAC density. An additional 3D variability analysis was conducted on the exit tunnel to remove particles with noisy artifacts. The final set of 36,986 particles with stable NAC and P-site tRNA density were polished using the reference-based motion correction package implemented in CryoSPARC. A homogenous refinement was conducted on the polished particles which yielded the HSP60 NAC-RNC map at a global resolution of 3.04 Å- determined by the gold standard Fourier Shell Correlation (FSC) of two half-sets of particles processed independently with an FSC threshold of 0.143. The local resolution of the map was calculated in CryoSPARC at an FSC threshold of 0.143.

Cryo-EM data processing strategy for OXA1L RNC and OXA1L RNC
Both OXA1L RNC and OXA1L RNC were processed in the same manner. Movies were motion and CTF corrected in CryoSPARC. Blob picker was used to pick particles with a diameter of 250-400 Å. Picked particles were extracted at a box size of 512 x 512 pixels and binned to 128 x 128 pixels for 2D classification. Ab-initio reconstruction was used to generate reference maps for 3D classification of all extracted particles to select 80S ribosome particles and exclude poorly aligning or junk particles. A homogenous refinement was conducted on the final set of 80S ribosome particles. Subsequent 3D variability analyses were conducted to sort for particles with stable P-site tRNA and NAC density using focused spherical masks. Particles were re-extracted at full pixel size and refined yielding high-resolution maps of ribosomes translating the OXA1L MTS and the MTS deletion mutant of OXA1L. The local resolution was calculated in CryoSPARC at an FSC threshold of 0.143.

Model Building
Following data processing, a model of the 80S ribosome (PDB 6R5Q) and NAC (PDB 7QWR) was docked into the cryo-EM map using ChimeraX54. NAC and eL22 were further fitted into the map as rigid bodies in Coot and manually adjusted based on side chain densities observed in the map. The unresolved N-terminal fluke of NACβ was built de novo guided by the visible side chain density. PHENIX55 was used to refine the model in the NAC-RNC density map with 3 macrocycles of real space refinements applying Ramachandran, sidechain rotamer, and protein secondary structure to correct for clashes. The final model was validated using MolProbity in PHENIX and figures were made in ChimeraX.

Isolation of Ribosomes from HEK293 Cells
Ribosomes were isolated from HEK293 GnTI cell pellets. Cell pellets were resuspended in lysis buffer (50 mM HEPES-KOH pH 7.7, 100 mM KOAc, 5 mM Mg(OAc)2, 0.5% IGEPAL, 1 mM DTT, 0.5X Protease inhibitor (Promega), 0.04 U/µL RNase inhibitor (Promega)) and lysed by passing the resuspension through a 23G x 1 ¼ needle four times (BD, cat no. 305120). The lysate was centrifuged at 11,000 x g for 10 min at 4°C. The supernatant was layered 1:1 on a sucrose cushion (50 mM HEPES-KOH pH 7.7, 750 mM KOAc, 5 mM Mg(OAc)2, 2 mM TCEP, 30% sucrose (w/v)) and centrifuged at 100,000 rpm for 1 hr at 4°C using a TLA 120.1 rotor. The pellets were resuspended in ribosome buffer (50 mM HEPES-KOH pH 7.7, 100 mM KOAc, 2 mM Mg(OAc)2, 2 mM TCEP, 0.5X protease inhibitor, 0.04 U/µL RNase inhibitor (Promega)). The concentration was determined on the Nanodrop (ThermoFisher) and aliquots of isolated ribosomes were snap frozen in liquid nitrogen.

NAC-Ribosome Co-sedimentation Assay
NAC variants and ribosomes were thawed on ice. Ribosomes were centrifuged at ~19,000 x g for 10 min at 4°C and the supernatant was transferred to a sterile 1.5 mL tube. NAC was diluted to 10 µM in reaction buffer (50 mM HEPES-KOH pH 7.7, 100 mM KOAc, 15 mM Mg(OAc)2, 0.04 U/µL RNase Inhibitor (Promega)). Ribosomes were diluted to 13 A260 units/mL in reaction buffer and NAC variants were added at a final concentration of 0.2 µM. The reaction was incubated at 30°C for 20 minutes and then moved to ice. A 50 µL portion of the reaction was layered on top of a sucrose cushion (50 mM HEPES-KOH pH 7.7, 100 mM KOAc, 15 mM Mg(OAc)2, 0.5X protease inhibitor (Promega), 0.04 U/µL RNase inhibitor (Promega), 25% sucrose (w/v)) and centrifuged at 100,000 rpm for 1 hr at 4°C using a TLA 120.1 rotor (Beckman). The pellet was resuspended in 1X SDS-PAGE loading dye (50 mM Tris-HCl pH 6.8, 2% SDS, 1% β-mercaptoethanol, 6% glycerol, 0.004% bromophenol blue) for analysis by Western Blot.

CRISPR/Cas9-based Knockout HEK293T cells
HEK293T cells, obtained from ATCC, were cultured at 37°C with 5% CO2 in DMEM with 10% fetal bovine serum (Fisher Scientific). Generation of NACβ-KO HEK293T cells was performed using CRISPR/Cas9. sgRNA oligonucleotide designed for human NACβ (5’-TGCTCGCAGAAAGAAGAAGG-3’) was inserted into lentiCRISPR, version 2 (Addgene 52961). Cells grown in 10 cm petri dishes were transfected with indicated plasmids using 5 μl 1 mg/ml polyethylenimine (PEI) (Millipore Sigma) per 1 μg of plasmids for HEK293T cells. The cells were cultured 24 hours after transfection in medium containing 2 μg/ml puromycin for 24 hours and then in normal growth medium. Single cell isolations were conducted to select for HEK293T cells with NACβ knocked out.

Thapsigargin (TG) treatment
HEK293T cells treated with 100 nM thapsigargin for 4 hours were included as positive controls for ER stress.

RNA preparation and Reverse Transcription PCR (RT-PCR)
XBP1 splicing was assessed in HEK293T cells as described previously56,57. Briefly, total RNA was extracted from cells using TRI Reagent and BCP phase separation reagent (Molecular Research Center, TR 118). RT-PCR primer sequences are:
hXBP1: F: 5’-GAATGAAGTGAGGCCAGTGG-3’ R: ACTGGGTCCTTCTGGGTAGA- 3’
hL32 F: 5’-AGTTCCTGGTCCACAACGTC-3’ R: 5’-TTGGGGTTGGTGACTCTGAT- 3’
h- denotes human genes.

Plasmids
Gene sequences encoding NACα and NACβ were synthesized and cloned into a pcDNA3.1(+)C-(K)-DYK vector (Genscript). QuickChange site-directed mutagenesis (Agilent, cat. no. 210518) was used to introduce two stop codons upstream of the C-terminal FLAG tag in NACβ, and to generate NAC switch mutants in which His34 and Arg35 or Lys42 and Phe46 were mutated to alanine (termed HR:AA and KF:AA, respectively).

NAC Rescue Experiments
NACβ-KO HEK293T cells grown in 3.5 or 10 cm petri dishes were transfected with 1 μg indicated plasmids with 5 μl 1mg/ml Polyethylenimine Max (PEI MAX) (Polysciences, 24765). Where indicated, cells were co-transfected with plasmids to express NACα and NACβ, or an empty vector. The cells were cultured for 24 hours after transfection, then pelleted and snap-frozen in liquid nitrogen for Western Blot or pulldown experiments. The proteins were extracted by sonication in NP-40 lysis buffer (50 mM Tris-HCl at pH7.5, 150 mM NaCl, 1% NP-40, 1 mM EDTA) with protease inhibitor (Millipore Sigma), DTT (Millipore Sigma, 1 mM), and phosphatase inhibitor cocktail (Millipore Sigma). Lysates were incubated on ice for 30 minutes and centrifuged at 16,000 x g for 10 minutes. Supernatants were collected and analyzed for protein concentration using Bio-Rad Protein Assay Dye (Bio-Rad). From 10 to 30 μg of protein was denatured at 95°C for 5 minutes in 5× SDS sample buffer (250 mM Tris-HCl pH 6.8, 10% sodium dodecyl sulfate, 0.05% bromophenol blue, 50% glycerol, and 1.44 M β-mercaptoethanol).

Glycoprotein Isolation by ConA Affinity Pulldown
ConA pulldown was performed using the Thermo Scientific Glycoprotein Isolation Kit (ThermoFisher, cat no. 89804) according to the manufacturer’s instructions with minor modifications. Briefly, the kit’s Binding/Wash Buffer was supplemented with 1% NP-40 and used as the lysis buffer. Frozen cell pellets were lysed in this buffer at 4°C in the presence of protease and phosphatase inhibitors. Lysates were centrifuged, and the supernatant was collected and diluted fourfold to reduce the NP-40 concentration. Approximately 3 mg of total protein was incubated with 50 µL ConA resin overnight at 4°C with gentle rocking. The resin was then washed four times with Binding/Wash Buffer, and bound glycoproteins were eluted with 100 µL Elution Buffer. Eluates were mixed with SDS sample buffer and analyzed by SDS-PAGE followed by immunoblotting.

Western Blot
Proteins were resolved by SDS–PAGE using either Bis-Tris gels (GenScript, cat. nos. M00653, M00654; Thermo Fisher, cat. no. NP0323BOX) run in MES buffer (GenScript, cat. no. M00677) or Tris-glycine gels, and then transferred to either 0.2 µm nitrocellulose membranes (LI-COR, cat. no. 926-31092) or PVDF membranes (Thermo Fisher). Membranes were blocked in 5% (w/v) milk in 1x PBST (0.1% Tween-20) for 1 h at room temperature, then incubated with primary antibodies diluted in 2% (w/v) BSA in 1x PBST. Primary antibodies used were: anti-FLAG (Sigma, F1804, 1:1,000), anti-RPL10a (Invitrogen, MLA5-44710, 1:2,000), anti-HSP90 (Santa Cruz, sc-13119, 1:5,000), anti-BiP (Abcam, ab21685, 1:5,000), anti-PDI (Enzo, ADI-SPA-890, 1:5,000), anti-NACα (Biorbyt, orb4116R71, 1:1,000), anti-NACβ (Invitrogen, PA5-63299, 1:2,000), and anti-CARL (Proteintech, 27298-1-AP, 1:5,000). Fluorescent secondary antibodies (anti-mouse, Invitrogen A21058; anti-rabbit, Invitrogen A32735, 1:10000) were detected using a LI-COR Odyssey imager. For chemiluminescent detection, HRP-conjugated secondary antibodies (Bio-Rad, 1:10,000) were incubated for 1 h at room temperature and developed using the ECL Chemiluminescence Detection System (Bio-Rad). Band intensities were quantified using Image Lab software (Bio-Rad, v6.1). Uncropped blots and gels are supplied in the source data file.

Data Availability
The cryo-EM maps and corresponding atomic models generated in this study have been deposited in the PDB and EMDB under accession code: EMD-48552 [https://www.ebi.ac.uk/emdb/EMD-48552] and PDB-9MR4 [https://doi.org/10.2210/pdb9mr4/pdb] for the OXA1L NAC-RNC MTS structure; EMD-71310 [https://www.ebi.ac.uk/emdb/EMD-71310] for HSP60 NAC-RNC ; EMD-71286 [https://www.ebi.ac.uk/emdb/EMD-71286] and EMD-71287 [https://www.ebi.ac.uk/emdb/EMD-71287] for the H59-undocked and -docked barrel states of the OXA1L structure, respectively. All data related to this study are available in the main text or the supplementary material. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The authors have revised the manuscript and added new data that alleviate some concerns that I and other reviewers had raised. While FRET and cryo-EM suggest that NAC is more stably bound in the presence of MTS rather than SS, the mechanistic aspects remain mysterious in the absence of the resolved interaction between MTS and NAC (see a suggestion below). Furthermore, the mechanistic roles of NAC for the three groups of proteins – mitochondria-targeted, ER-targeted, and cytoplasmic – are not easily reconcilable, as currently presented in the manuscript and detailed below.\nIn the revised manuscript, the most stressed is the point that NAC must help distinguish mitochondria-targeted proteins from ER-targeted proteins. This point is supported by the observations that NAC does not stably interact with the hydrophobic SS allowing the latter to interact with SRP; the authors propose that in contrast to SS, MTS sequences interact with NAC, so the latter shields them from SRP. An important second point, following this logic, is whether NAC also protects cytosolic (or non-MTS/non-SS) proteins from SRP – here the authors come to the opposite conclusion. To address this point, FRET and cryo-EM experiments were done, comparing Oxa1L-MTS and Oxa1L-deltaMTS constructs. For FRET experiments, a few minor technical clarifications would be helpful (see below). As for high-resolution cryo-EM maps (shown in Extended Figures, up to Ext. Fig. 12), the authors conclude that NAC is more stable in the Oxa1L-MTS map than in the Oxa1L-deltaMTS map. The maps, however, appear to show similar binding modes of NAC, as they both feature bh2 and the b-barrel. These are less well resolved in the Oxa1L-deltaMTS map (Ext. Fig. 12), but the global resolution of this map also appears to be visibly lower (compare 80S-WT reconstruction in Ext. Fig. 3C – with ribosomal nucleotides resolved and local resolution achieving 2 A – and 80S-deltaMTS in Ext. Fig. 11C – with overall lower-resolution 80S and no local resolution shown). Lower-resolution peripheral features therefore do not provide a strong support for the difference, unless additional analyses show otherwise. Second, the lower-resolution classes for the deltaMTS complex (Fig. 4F) suggest NAC barrel mobility, however it is unclear if the same classification approach was applied to the MTS complex; low-resolution heterogeneity in the resulting classes must be shown and discussed. To conclude this topic, the authors’ premise that NAC differently interacts with MTS and non-MTS (cytosolic) sequences (1) needs additional support as suggested above; and (2) creates a paradox - if NAC stabilization protects MTS from promiscuous interactions with the SRP – how does it similarly protect cytoplasmic proteins, which according to these conclusions, do not stabilize NAC?\nThe conclusion that NAC stabilization by MTS plays a specific role in the fate of MTS-containing proteins remains a concern, in the absence of the structural mechanism of specific stabilization. Have the authors attempted to predict MTS-NAC interactions in Alphafold 3 then search for the predicted contacts in low-pass-filtered maps? This important issue – how an unobserved interaction might explain specificity - needs to be further elaborated in discussion.\nA few technical clarifications: to better understand whether FRET differences between WT and delta-MTS constructs are caused by NAC mobility rather than by other effects (e.g. different dye positions), it would be helpful to locate the dye on Oxa1L constructs (at residue 39) relative to NAC:\na. Update the construct scheme (Fig. 4A) to illustrate the positions of aa39, tRNA and schematic PET boundaries.\nb. Similarly, specify the position of aa39 in the tunnel on the Oxa1L structure or map in one of the figures or an additional panel.\nc. Does the amphiphilic helix of Oxa1L deletion in the delta-MTS mutant change the position of the dye relative to NAC? Like above, show the dye position within the deltaMTS map (in a main or supplementary figure).\nAndrei Korostelev", "reviewer_4": "The authors have addressed my comments with additional experiments and I support publication of this study.", "reviewer_5": "The study by Maldosevic and colleagues presents a novel structure of the mammalian ribosome bearing a mitochondrial targeting sequence in complex with NAC. Surprisingly this reveals a novel binding mode for NAC where in addition to the interaction via the canonical ribosome anchor in βNAC a contact is formed with h59 causing its repositioning. This binding mode of NAC is incompatible with functional binding of SRP to the ribosome and so could provide a mechanism to prevent mistargeting of a MTS by SRP. I did not review the first version of the MS, where several reviewers quite reasonably questioned, firstly, whether this unexpected binding mode was specific for a mitochondrial targeting signal and secondly how this was rationalised mechanistically considering that the nascent chain appears to not be stably bound to NAC in this complex at least to the extent that it can be observed in the structure. The revised manuscript is much improved and addresses many of the original criticisms and also now has a more focussed message.\nIn the revised manuscript, the authors have determined additional structures using a more stable nascent chain by using the XBP1 stalling peptide and a similar nascent chain that lacking the MTS. This is complemented with FRET experiments to assess the dynamics of NAC and SRP with a nascent chain containing the MTS or the MTS deleted or replaced with a signal peptide. These experiments strengthen the original findings as they show that rearrangement and stable association to h59 is specific for the presence of the MTS. However, despite the more stable nascent chain and visible density for the nascent chain in the exit tunnel, there is still no visible association of the MTS with NAC which might explain the stabilised h59 binding. Would the authors envisage that transient dynamic binding of the MTS to NAC is able cause specific rearrangements, which promote the capture and repositioning h59, which can then persist even in after the MTS dissociates? It should be noted that the assembly of the complex here is slightly non-physiological as purified NAC is added to salt washed stalled nascent chain, rather than being present during elongation where it might have initially been captured at a shorter chain length.\nIt is intriguing that an MTS, but not a signal peptide nor simply a nascent chain without the MTS triggers, this rearrangement to avoid SRP mistargeting. Is that because a specific mechanism is required as an MTS is specifically more prone to mistargeting and/or reflects an alternate binding state that feeds into the mitochondrial targeting machinery?\nConsistent with a role of NAC in preventing mistargeting of mitochondrial proteins, the authors now show that in a βNAC ko line, neuclear-encoded mitochondrial proteins are mislocalised. The inference is that they are targeted to the ER and the mild UPR induction would be consistent with this as would previous observations from the Deuerling group in C. elegans. Can this be shown directly eg by repeating the IF with an ER marker in the β-NAC ko line, or better still in the ko line expressing the switch mutations? The latter would more specifically link the effects in vivo to the proposed switch mechanism. I wasn’t completely convinced by the authors assertion in their rebuttal that co-localisation with ER markers could not be done, as this was nicely demonstrated in other studies such as the missorting of TA mitochondria proteins to the ER (McKenna et al [2020] Science 369: eabc5809). An alternative would be to detect N-glycosylation of mitochondria proteins (assuming they have a NXS/T motif) which could also confirm mis-localisation. This analysis would greatly strengthen the model and principal message that the NAC switch provides a mechanism to prevent mistargeting of MTS-containing proteins to the ER by SRP.\nMinor points:\nIn the overview of the NAC expression construct (Extended data Fig. 2B) I assume the RBS should come before the cleavable HIS tag?\nI found extended figure 5C slightly confusing. Why is the purple overlay only present in the top left panel?\nIn the FRET figure can the position of the nascent chain probes be more clearly indicated eg in the schematic as in figure 4A. The position of the NAC FRET probe could also be shown in Figure 3A.\nAlso does the positioning of the nascent chain in the tunnel change upon remove of the MTS? Compaction of the nascent chain for example could alter the positioning of the nascent chain FRET probe which would alter the interpretation."}, "author_response": "We thank the reviewers for their thorough and constructive review of our revised manuscript. Additional\nexperiments and changes to the text were made to address reviewer comments. The revisions of the main text and\nsupplementary information are highlighted in yellow and the detailed responses to comments are provided below.\nReviewer #1 (Remarks to Author)\nComment: 1) To conclude this topic, the authors’ premise that NAC differently interacts with MTS and non-MTS\n(cytosolic) sequences (1) needs additional support as suggested above; and (2) creates a paradox - if NAC stabilization\nprotects MTS from promiscuous interactions with the SRP – how does it similarly protect cytoplasmic proteins, which\naccording to these conclusions, do not stabilize NAC?\nAuthor response: We thank the reviewer for the thoughtful and constructive comments. NAC is known to support\nboth cytosolic and mitochondrial protein biogenesis, and its depletion broadly mistargets ribosomes to the ER\n(PMID: 25859040). Our study focuses on NAC engagement with ribosomes translating mitochondrial targeting\nsequence (MTS) bearing nascent chains, and our analyses are therefore specific to this subset of translating\nribosomes. We do not consider an MTS-deletion construct to be an adequate mimic of a bona fide cytosolic\nnascent chain, as distinct N-terminal features and early folding events in cytosolic proteins likely modulate NAC\nengagement. Therefore, addressing how NAC interacts with cytosolic nascent chains is beyond the scope of the\ncurrent manuscript.\nWe also note that an MTS shares more similarity to ER signal sequences compared to cytosolic proteins: the\nformer contains an N-terminal amphiphilic helix (or helices) harboring a combination of hydrophobic and basic\nresidues, and the latter is an N-terminal hydrophobic helix flanked by basic residues. Neither of these features are\nfound at the N-terminus of most cytosolic proteins. The relationship of ER signal sequence vs MTS and cytosolic\nproteins is analogous to that of cognate vs near-cognate and non-cognate substrates: the former requires more\nelaborate mechanisms for discrimination by SRP compared to the latter. Nevertheless, we agree that how NAC\ninteracts with cytosolic nascent chains remains an important open question, and we have expanded the Discussion\nsection to explicitly note this limitation in the current study.\nDiscussion, lines “328-330”: “While our study focuses on elucidating the molecular mechanism by which NAC\nprevents SRP from accessing mitochondrial nascent chains via a molecular switch, how NAC engages with\ncytonuclear clients remains unresolved and requires further investigation”.\nComment: 2) As for high-resolution cryo-EM maps (shown in Extended Figures, up to Ext. Fig. 12), the authors\nconclude that NAC is more stable in the Oxa1L-MTS map than in the Oxa1L-deltaMTS map. The maps, however,\nappear to show similar binding modes of NAC, as they both feature bh2 and the b-barrel. These are less well\nresolved in the Oxa1L-deltaMTS map (Ext. Fig. 12), but the global resolution of this map also appears to be\nvisibly lower (compare 80S-WT reconstruction in Ext. Fig. 3C – with ribosomal nucleotides resolved and local\nresolution achieving 2 A – and 80S-deltaMTS in Ext. Fig. 11C – with overall lower-resolution 80S and no local\nresolution shown). Lower-resolution peripheral features therefore do not provide a strong support for the\ndifference, unless additional analyses show otherwise.\nAuthor response: In the revised work, our interpretation of NAC stability in the presence or absence of the MTS\nis primarily investigated by smTIRFM analyses of β-barrel dynamics across different nascent-chain contexts and\nNAC mutants on the ribosomes. The cryo-EM analysis of the OXA1L-∆MTS was performed as supportive\nstructural evidence. While similar barrel–ribosome contacts are observed, a 3D variability analysis indicated that\na subset of particles populates conformations with greater NAC barrel and H59 movement, which suggests\nincreased dynamics upon MTS deletion. However, our single-particle cryo-EM cannot resolve dynamic behavior\nover the timescales accessible by smTIRFM. Thus, the lower resolution of NAC density observed in the OXA1L-\nΔMTS map is therefore consistent with, but not the sole basis for our functional conclusions.\nFor clarity, the two states have been renamed to H59-docked\nand -undocked to emphasize the functional conclusions with\nour smTIRFM assays instead. Additionally, Supplementary\nFig. 13 (map comparisons) has been simplified, and\nSupplementary Fig. 11 has been updated to include local-\nresolution estimates (Response Figure 1A–B).\nResponse Figure 1. Local resolution of cryo-EM maps from\nthe different NAC barrel states related to Supplementary Fig.\n11. (A-B) Maps for the H59-undocked and -docked state\ncolored by local resolution estimates from CryoSPARC at\nan FSC cutoff of 0.143. Insets show close-up views of the\nNAC barrel corresponding to the red boxed regions of the\nmap.\nComment: 3) Second, the lower-resolution classes for the deltaMTS complex (Fig. 4F) suggest NAC barrel\nmobility, however it is unclear if the same classification approach was applied to the MTS complex; low-\nresolution heterogeneity in the resulting classes must be shown and discussed.\nAuthor response: We applied the same 3D classification approach to the MTS dataset using identical masks and\nparameters in CryoSPARC. Although barrel dynamics are detectable in the MTS data, the combination of a\nlowered NAC barrel state coupled to H59 conformational change was only observed in the ΔMTS dataset (Class\n3, Response Fig. 2A–B). As noted above (Reviewer 1, Comment 2), we have removed interpretation of barrel\nstability based on peripheral map features in the revised manuscript. We have added Supplementary Fig. 12\nreporting the 3D variability analysis for both datasets and updated the Results to discuss low-resolution\nheterogeneity. Please see updated lines “230-234” in the Results section titled “MTS mutations destabilize the\nNAC barrel on the ribosome.”\nResponse Figure 2. 3D variability analysis of OXA1L and OXA1L particles. (A-B) Processing scheme\nMTS ∆MTS\nfor the OXA1L NAC-RNC dataset (left) and the OXA1L NAC-RNC dataset (right). Classes with clear\nMTS ∆MTS\nbarrel occupancy are depicted below each processing scheme. The NAC-RNC model is fitted in each map for\nMTS\nreference. Maps are shown as transparent surface, filtered to 10Å for visualization.\nComment: 4) The conclusion that NAC stabilization by MTS plays a specific role in the fate of MTS-containing\nproteins remains a concern, in the absence of the structural mechanism of specific stabilization. Have the authors\nattempted to predict MTS-NAC interactions in Alphafold 3 then search for the predicted contacts in low-pass-\nfiltered maps? This important issue – how an unobserved interaction might explain specificity - needs to be further\nelaborated in discussion.\nAuthor response: This is an excellent and important point. Based on our results, this likely reflects either a\ntransient interaction or an allosteric effect triggered by the MTS that influences the conformation of ribosomal\nH59 and the specificity of NAC binding. AlphaFold 3.0 multimer predicts a potential interaction between the\nOXA1L MTS and the NAC β-barrel (Response Fig. 3A-B). However, inspection of the cryo-EM map does not\nreveal convincing density for the MTS at this position, and at higher contour levels only weak peripheral features\nare visible that cannot be unambiguously assigned to the MTS (Response Fig. 3C).\nTo elaborate on this limitation, we added a statement in the discussion based on these results, lines “307-309”:\n“The absence of discernible density for the MTS in our cryo-EM maps suggests that its interaction with NAC is\neither highly transient or mediated through an allosteric effect that modulates NAC conformation and ribosome\nengagement.”\nResponse Figure 3. Alphafold prediction of MTS interactions with the NAC barrel. (A) Alphafold 3.0 model of\nthe OXA1L MTS-NAC interaction aligned to the NAC barrel from our experimental NAC-RNC structure.\nMTS\n(B) Predicted aligned error plot for the Alphafold prediction in (A) where regions of high confidence and low\nconfidence are represented by dark and light green shading, respectively. (C) Overlay of the NAC barrel density\nat lower contour with the model of the MTS. The EM map is depicted as a transparent surface and was low-pass\nfiltered to 8Å for visualization.\nComment: 5) A few technical clarifications: to better understand whether FRET differences between WT and\ndelta-MTS constructs are caused by NAC mobility rather than by other effects (e.g. different dye positions), it\nwould be helpful to locate the dye on Oxa1L constructs (at residue 39) relative to NAC:\n(a) Update the construct scheme (Fig. 4A) to illustrate the positions of aa39, tRNA and schematic PET boundaries.\nAuthor response: Across all constructs, the amber codon for dye labeling is\n35 amino acids from the C-terminus of the nascent chain, which positions\nthe dye at the opening of the ribosome exit tunnel. We have updated our\nconstruct schematic in Fig. 4A and the accompanying legend to clarify this\n(red star, Response Fig. 4).\nResponse Figure 4. Construct schematics for the nascent-chain variants\nused in Fig. 4A. OXA1L variants in which the N-terminal amphipathic helix\n(residues 4–21, “aMTS”, dark blue) was either deleted (ΔMTS) or replaced\nwith the hydrophobic core of the preprolactin ER signal sequence (“SS,”\ngreen). The FRET acceptor was positioned at 35 amino acids from the C-\nterminus in all nascent chains tested (red star). The gray box denotes the\napproximate span of residues located within the ribosomal exit tunnel.\n(b) Similarly, specify the position of aa39 in the tunnel on the Oxa1L structure or map in one of the figures or an\nadditional panel.\nAuthor response: Outside the exit tunnel, the nascent chain becomes too flexible for reliable modeling beyond\nresidue 42. The FRET acceptor is positioned at Leu39 in the OXA1L-MTS structure, within 2 residues of the\nmodeled N-terminal amino acid. In an extended conformation, 2 amino acids would lengthen the nascent chain\nby ~7 Å. A mask with a radius of 7 Å centered on the terminal residue places the acceptor near the tunnel opening\nand within proximity for FRET with the donor on the NAC barrel (Response Fig. 5).\nResponse Figure 5. Relative\npositioning of the NAC FRET\ndonor and nascent-chain\nFRET acceptor. (A) Model\nshowing NAC docked at the\ntunnel exit with the donor dye\nindicated (red sphere). A 7 Å\nradius mask (purple) centered\non the terminal modeled\nresidue of the nascent chain\napproximates the position of\nthe FRET acceptor on the nascent chain near the tunnel opening. (B) Updated Fig. 3A panel indicating the position\nof the FRET donor and approximate location of the FRET acceptor in the NAC-RNC model.\nMTS\n(c) Does the amphiphilic helix of Oxa1L deletion in the delta-MTS mutant change the position of the dye relative\nto NAC? Like above, show the dye position within the deltaMTS map (in a main or supplementary figure).\nAuthor response: Regardless of the mutations we introduced into the MTS, the FRET acceptor is ~35 aa from the\nC-terminus of the nascent chain across all constructs tested (reviewer 1, comment 6a). Additionally, our maps did\nnot show differences in nascent chain compaction in the tunnel that could influence FRET readouts between the\nexperiments (Response Fig. 6).\nResponse Figure 6. Nascent chain density in the MTS and ∆MTS maps. Close-ups of the nascent chain in the\nexit tunnel for the three maps relative to NAC. Maps were low-pass filtered to 6Å for visualization.\nReviewer #2 (Remarks to the Author):\nComment: 1) The authors have addressed my comments with additional experiments and I support publication\nof this study.\nAuthor response: We thank the reviewer for their positive assessment of our revised manuscript and their support\nfor publication.\nReviewer #4 (Remarks to the Author):\nComment: 1) The study by Maldosevic and colleagues presents a novel structure of the mammalian ribosome\nbearing a mitochondrial targeting sequence in complex with NAC. Surprisingly this reveals a novel binding mode\nfor NAC where in addition to the interaction via the canonical ribosome anchor in βNAC a contact is formed\nwith h59 causing its repositioning. This binding mode of NAC is incompatible with functional binding of SRP to the\nribosome and so could provide a mechanism to prevent mistargeting of a MTS by SRP. I did not review the first\nversion of the MS, where several reviewers quite reasonably questioned, firstly, whether this unexpected binding\nmode was specific for a mitochondrial targeting signal and secondly how this was rationalised mechanistically\nconsidering that the nascent chain appears to not be stably bound to NAC in this complex at least to the extent\nthat it can be observed in the structure. The revised manuscript is much improved and addresses many of the\noriginal criticisms and also now has a more focused message.\nAuthor response: We thank the reviewer for their positive comments and effort in reviewing both our original and\nrevised manuscript and we appreciate the recognition that the study presents a novel NAC binding mode that\nprevents mistargeting of mitochondrial proteins to the ER.\nComment: 2) In the revised manuscript, the authors have determined additional structures using a more stable\nnascent chain by using the XBP1 stalling peptide and a similar nascent chain that lacking the MTS. This is\ncomplemented with FRET experiments to assess the dynamics of NAC and SRP with a nascent chain containing\nthe MTS or the MTS deleted or replaced with a signal peptide. These experiments strengthen the original findings\nas they show that rearrangement and stable association to h59 is specific for the presence of the MTS. However,\ndespite the more stable nascent chain and visible density for the nascent chain in the exit tunnel, there is still no\nvisible association of the MTS with NAC which might explain the stabilised h59 binding. Would the authors\nenvisage that transient dynamic binding of the MTS to NAC is able cause specific rearrangements, which promote\nthe capture and repositioning h59, which can then persist even in after the MTS dissociates?\nAuthor response: We appreciate this insightful question. As noted in our response to Reviewer 1, the absence of\ndiscernible MTS density in the cryo-EM map suggests that its interaction with NAC is likely dynamic and/or that\nthe MTS may act via an allosteric effect on the ribosome. We agree that such a dynamic interaction could promote\nthe observed H59-docked state stabilizing the NAC β-barrel, even after the MTS disengages. Given that the\nnascent chain continues to evolve during translation elongation, it is also plausible that folded domains emerging\nfrom the ribosome later in translation could alter the H59 conformation and displace the NAC barrel from the\nribosome surface. A statement on the potential transient MTS interaction with NAC has been explicitly mentioned\nin the revised manuscript (Discussion, lines 328–330).\nComment: 3) It should be noted that the assembly of the complex here is slightly non-physiological as purified\nNAC is added to salt washed stalled nascent chain, rather than being present during elongation where it might\nhave initially been captured at a shorter chain length.\nThis approach was chosen to obtain a homogeneous population of ribosomes suitable for high-resolution\nstructural analysis. Similar reconstitution strategies have proven highly effective for studying ER-targeting\nmechanisms and, more recently, nascent-chain processing events (PMID: 35201867, PMID: 37347872, PMID: 39169182, PMID: 40639378, PMID: 40859030).\nWe have added a statement in the results (lines 109-111) describing the assembly of the complex with this noted\nlimitation: “NAC was added to salt-washed stalled ribosome–nascent chain complexes (RNCs), exposing the N-\nterminal MTS (RNC ) of OXA1L (74 residues) or HSP60 (62 residues), to obtain a homogeneous population\nMTS\nsuitable for structural analysis.”\nComment: 4) It is intriguing that an MTS, but not a signal peptide nor simply a nascent chain without the MTS\ntriggers, this rearrangement to avoid SRP mistargeting. Is that because a specific mechanism is required as an\nMTS is specifically more prone to mistargeting and/or reflects an alternate binding state that feeds into the\nmitochondrial targeting machinery?\nAuthor response: We speculate that MTS-bearing nascent chains are particularly prone to mistargeting by SRP\ndue to their hydrophobicity and the presence of transmembrane domains in mitochondrial inner-membrane\nproteins. Therefore, a specific mechanism is required to ensure their correct delivery to mitochondria. Our cryo-\nEM and smTIRFM experiments indicate that the stabilized NAC-ribosome interaction serves to prevent premature\nSRP engagement at the ER. Nevertheless, an additional factor may be required to guide these nascent chains to\nthe mitochondrial import machinery, and the identity of this factor remains to be elucidated.\nComment: 5) Consistent with a role of NAC in preventing mistargeting of mitochondrial proteins, the authors\nnow show that in a βNAC ko line, nuclear-encoded mitochondrial proteins are mislocalised. The inference is that\nthey are targeted to the ER and the mild UPR induction would be consistent with this as would previous\nobservations from the Deuerling group in C. elegans. Can this be shown directly eg by repeating the IF with an\nER marker in the β-NAC ko line, or better still in the ko line expressing the switch mutations? The latter would\nmore specifically link the effects in vivo to the proposed switch mechanism. I wasn’t completely convinced by\nthe authors assertion in their rebuttal that co-localisation with ER markers could not be done, as this was nicely\ndemonstrated in other studies such as the missorting of TA mitochondria proteins to the ER (McKenna et al [2020]\nScience 369: eabc5809). An alternative would be to detect N-glycosylation of mitochondria proteins (assuming\nthey have a NXS/T motif) which could also confirm mis-localisation. This analysis would greatly strengthen the\nmodel and principal message that the NAC switch provides a mechanism to prevent mistargeting of MTS-\ncontaining proteins to the ER by SRP.\nWe thank the reviewer for the helpful suggestion to assess mistargeting of mitochondrial proteins via N-\nglycosylation. Since mitochondrial HSP60 contains a putative NXS/T glycosylation motif and prior work in C.\nelegans reported elevated glycosylated HSP60 in NACβ-KO lysates (PMID: 25859040), we also performed ConA\npulldowns using the human cell lysates of NACβ-KO reported in our study. The results from these experiments\nconsistently revealed increased glycosylated HSP60 in both NACβ-KO clones (Response Fig. 7A). Rescue\nexperiments showed partial recovery in WT complementation, but not with the NAC switch mutants (Response\nFig. 7B), consistently as observed in our ER stress experiments in Fig. 5. Together, these data further support our\nmodel that the NACβ switch prevents mistargeting of HSP60 and other MTS-containing mitochondrial proteins\nto the ER.\nResponse Figure 7. NACβ knockout\nleads to mistargeting of\nmitochondrial HSP60 to the ER. A)\nConcanavalin A (ConA) pull-down\nof mitochondrial HSP60 from WT\nand NACβ-KO HEK293T cell\nlysates. B) ConA pulldown of HSP60\nfrom cells transfected with WT or\nmutant NACβ plasmids. Band\nintensity for HSP60 was quantified\nrelative to control cells transfected\nwith an empty vector (EV).\nThese new results are now added in\nSupplementary Fig. 15 in the revised\nmanuscript.\nComment: 6) In the overview of the NAC expression construct (Extended data Fig. 2B) I assume the RBS should\ncome before the cleavable HIS tag?\nAuthor response: Thank you for spotting this error, we have adjusted our NAC expression construct schematic to\ncorrect the positioning of the RBS (Response Fig. 8).\nResponse Figure 8. NAC expression construct schematic with correction made to the RBS positioning.\nSchematics created in BioRender. Maldosevic, E. (2026) https://BioRender.com/3wi39a1.\nComment: 7) I found extended figure 5C slightly confusing. Why is the purple overlay only present in the top\nleft panel?\nAuthor response: We have revised Supplementary\nFig. 5C by removing the model fits, simplifying the\npanel and focusing the comparison on the\nsimilarity between the two maps. We now show\ntwo different views for the maps (Response Fig. 9).\nResponse Figure 9. Overlay of the OXA1L and\nHSP60 RNC cryo-EM maps related to\nMTS\nSupplementary Fig. 5. Close-up views of the NAC\nbarrel docking site on the ribosome depicting the\noverlap between the OXA1L and HSP60 RNCs.\nComment: 8) In the FRET figure can the position of the nascent chain probes be more clearly indicated eg in the\nschematic as in figure 4A. The position of the NAC FRET probe could also be shown in Figure 3A. Also does\nthe positioning of the nascent chain in the tunnel change upon remove of the MTS? Compaction of the nascent\nchain for example could alter the positioning of the nascent chain FRET probe which would alter the\ninterpretation.\nAuthor response: In the revised manuscript, we have clearly labeled the FRET probes in both Fig. 3A and Fig.\n4A. Additionally, we did not see differences in nascent chain compaction between the different structures\ndetermined. Please refer to reviewer 1, comments 5a-c for additional information regarding these important\npoints."}, {"round": 2, "reviewer_comments": {"reviewer_1": "My technical criticisms have been addressed, and the manuscript has improved. The absence of MTS-NAC interaction, however, remains perplexing in the light of the proposed mechanistic model. Have the authors considered a model, in which rather than invisibly interacting with NAC, MTS prevents NAC displacement from the ribosome by NOT interacting with it (as opposed to ER or deltaMTS sequences)? This alternative scenario may be worth mentioning in Discussion, where the absence of this interaction is discussed. If correct, NAC would be predominantly in the H59-docked conformation in the absence of any nascent peptide at PET exit (e.g., a short peptide) - if such/similar data exist, the authors could bring them up to argue that the alternative scenario is more/less likely than transient/allosteric interactions.\nAndrei Korostelev", "reviewer_5": "The authors have satisfactorily addressed the majority of the issues I had raised improving the manuscript further. The only outstanding issue relates to the new data in Supp. Fig. 15. The authors, as suggested, have used the N-glycosylation of mitochondrial Hsp60 to monitor mislocalisation of mitochondrial proteins to the ER and can confirm that the β-NAC ko does indeed lead to such mis-localisation (as previously reported). However, the rescue experiments to specifically test the role of the NAC switch mechanism in this process using the β-NAC mutants were less compelling. The WT rescue construct leads to very strong over-expression of β-NAC, much more than was observed in the previous rescue experiments (fig. 5), yet this leads to only a modest (30%) reduction in Hsp60 mislocalisation, which appears less effective in the switch mutants. This was a much smaller rescue as compared to the UPR induction phenotype in the same system. Given the small size of the effects, a more robust quantification and statistical analysis (as shown in figure 5 and supp. fig 14) is required to make this data convincing. Also, in this experiment are α and β-NAC over-expressed from the same plasmid or separate ones? – the over-expression plasmid details are somewhat vague in methods section. Expression from the same plasmid would ensure that inefficient rescue is not due to a cohort of cells transfected with a single plasmid and hence only over-expressing one of the two subunits but not the other. Also reference to α-NAC over-expression is needed in the supp. fig 15 legend (as in fig. 5)."}, "author_response": "We thank the reviewers for their thorough and constructive review of our revised manuscript. Additional\nexperiments and changes to the text were made to address reviewer comments. The revisions of the main text and\nare highlighted in yellow and the detailed responses to comments are provided below.\nReviewer #1 (Remarks to Author)\nComment: 1) My technical criticisms have been addressed, and the manuscript has improved. The absence of MTS-NAC\ninteraction, however, remains perplexing in the light of the proposed mechanistic model. Have the authors considered a\nmodel, in which rather than invisibly interacting with NAC, MTS prevents NAC displacement from the ribosome by NOT\ninteracting with it (as opposed to ER or deltaMTS sequences)? This alternative scenario may be worth mentioning in\nDiscussion, where the absence of this interaction is discussed. If correct, NAC would be predominantly in the H59-docked\nconformation in the absence of any nascent peptide at PET exit (e.g., a short peptide) - if such/similar data exist, the authors\ncould bring them up to argue that the alternative scenario is more/less likely than transient/allosteric interactions.\nAuthor response: We agree with the reviewer that the manuscript would benefit from further discussion of a model\nwhere NAC barrel docking is maintained by H59. This docking modality can then be disrupted by emerging\nnascent chains that potentially interact with NAC the barrel domain.\nPlease see the updated discussion, lines 310-314, page 14 in the revised manuscript:\n“An alternative model is that NAC is displaced from the H59-docked conformation on ribosomes translating\ncytosolic or ER proteins due to direct interactions with the nascent chain. While our study focuses on elucidating\nthe molecular mechanism by which NAC prevents SRP from accessing mitochondrial nascent chains via a\nmolecular switch, how NAC engages with cytonuclear clients remains unresolved and requires further\ninvestigation.”\nReviewer #5 (Remarks to the Author):\nComment: 1) The authors have satisfactorily addressed the majority of the issues I had raised improving the\nmanuscript further. The only outstanding issue relates to the new data in Supp. Fig. 15. The authors, as suggested,\nhave used the N-glycosylation of mitochondrial Hsp60 to monitor mislocalisation of mitochondrial proteins to\nthe ER and can confirm that the β-NAC ko does indeed lead to such mis-localisation (as previously reported).\nHowever, the rescue experiments to specifically test the role of the NAC switch mechanism in this process using\nthe β-NAC mutants were less compelling. The WT rescue construct leads to very strong over-expression of β-\nNAC, much more than was observed in the previous rescue experiments (fig. 5), yet this leads to only a modest\n(30%) reduction in Hsp60 mislocalisation, which appears less effective in the switch mutants. This was a much\nsmaller rescue as compared to the UPR induction phenotype in the same system. Given the small size of the\neffects, a more robust quantification and statistical analysis (as shown in figure 5 and supp. fig 14) is required to\nmake this data convincing. Also, in this experiment are α and β-NAC over-expressed from the same plasmid or\nseparate ones? – the over-expression plasmid details are somewhat vague in methods section. Expression from\nthe same plasmid would ensure that inefficient rescue is not due to a cohort of cells transfected with a single\nplasmid and hence only over-expressing one of the two subunits but not the other. Also reference to α-NAC over-\nexpression is needed in the supp. fig 15 legend (as in fig. 5).\nAuthor response: We thank the reviewer for their suggestion. We repeated the NAC rescue experiment using an\nindependent NACβ-KO clone. In this experiment, we observe a stronger rescue by WT NAC to a level similar to\nthat observed in WT HEK293T cells (0.3 for WT versus 0.5 for rescue), while both NACβ mutants fail to rescue\nto the same extent (1.4 for the HR mutant and 0.8 for the KF mutant). Therefore, these data, together with our\nprevious results presented in Fig. 5, strengthen our conclusions regarding the role of the NAC molecular switch\nin preventing Hsp60 mislocalization. We also updated the Supplementary Fig. 15 legend to explicitly indicate\nNACα co-expression and revised the Methods accordingly to describe the co-expression strategy.\nPlease see the updated Supplementary Fig. 15 below, as referenced on page 16 of the Supplementary Data.\nResponse Figure 1. NACβ knockout leads to mistargeting of mitochondrial HSP60 to the ER. A) Concanavalin\nA (ConA) pulldown of mitochondrial HSP60 from WT and NACβ-KO HEK293T cell lysates. B) ConA pulldown\nof HSP60 from cells co-transfected with NACβ variants and WT NACα or an empty vector. Band intensity for\nHSP60 was quantified relative to control cells transfected with an empty vector (EV) and normalized to the\nloading control, CARL."}]

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