Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB (2022)
Figure 6

Fig. 6 PI4KB is an ARL5A and ARL5B interacting protein

(A) PI4KB is enriched by ARL5A and ARL5B. ARL5A, ARL5B, and RAB14 rank the top 3 GTPases based on PI4KB enrichment by miniTurbo screening of the GTP-bound forms of the Ras superfamily. For ARF family, data were from two biologically independent analyses while for other GTPases data were from one biological sample.

(B) Interaction of PI4KB with ARL5A/5B is stronger than ARF1, RAB11A/11B, and RAB14. Flag-tagged PI4KB was co-expressed with HA-tagged GTP-bound mutant of GTPases in HEK293A cells as indicated. PI4KB was immunoprecipitated with Flag beads, and the co-precipitated GTPases were detected by HA Western. The result is a representative of three biologically independent experiments.

(C) PI4KB preferentially binds to the GTP-form of ARL5B. Flag-tagged PI4KB was co-expressed with HA-tagged ARL5BQ70L (GTP-form) or ARL5BT30N (GDP-form) mutant in HEK293A cells. Interaction was measured similar to panel B. The result is a representative of two biologically independent experiments.

(D) The ARL5A colocalization with PI4KB is GTP-dependent. HA-tagged GTP-bound ARL5AQ70L, wild-type ARL5A or GDP-bound ARL5AT30N was co-expressed with Flag-tagged PI4KB. After 24 h, cells were fixed, followed by incubation with anti-Flag and anti-HA primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 10 μm. Shown is a representative of two biologically independent experiments. The left panel is the quantification of overlap coefficient. Mean± SD; n= 6 technically independent samples. One-way ANOVA; *P < 0.05.

(E) ARL5A/5B double knockout diminishes the colocalization of PI4KB and TGN46. Flag-tagged PI4KB was expressed in HEK293A wild-type or two ARL5A/5B double knockout clones as indicated. After 24 hours, cells were fixed, followed by incubation with anti-Flag and anti-TGN46 primary antibodies. DAPI (blue) was used for DNA staining. Scale bars: 5 μm. The result is a representative of two biologically independent experiments. The left panel is the quantification of overlap coefficient. Mean± SD; n=6 technically independent samples. One-way ANOVA; *P < 0.05.

(F) Schematic representation of the full-length PI4KB and various truncations. ∆N, deletion of the N-terminal region (1-306aa); ∆C, deletion of the C-terminal kinase domain (307-801aa); ∆N1, deletion of the N-terminal disordered region (1-127aa); ∆N2, deletion of the helical domain (128-242aa); ∆N3, deletion of the helical-kinase linker region(243-306aa). " + " denotes positive interaction with ARL5B and " - " denotes no interaction.

(G) The N1 region of PI4KB is required for the interaction with ARL5B. Flag-tagged full-length or truncations of PI4KB were co-expressed with HA-tagged ARL5BQ70L. PI4KB was immunoprecipitated with Flag beads, and the co-precipitated ARL5B was detected by HA Western. The result is a representative of three biologically independent experiments.

(H) The N1 region of PI4KB is required for the co-localization of ARL5A/B and PI4P. P4Mx2-mEGFP was co-expressed with HA-tagged ARL5AQ70L in HEK293A wild-type or PI4KB knockout cells (clone#2-5). HEK293A cells were co-transfected with indicated constructs. After 24 h, cells were fixed with 4% paraformaldehyde, followed by incubation with anti-HA primary antibodies. DAPI (blue) was used for nuclei staining. Scale bars: 10 μm. The result is a representative of two biologically independent experiments.

(I) Mander's overlap coefficient of (H). Mean± SD; n= 8 technically independent samples. One-way ANOVA; *P < 0.05.