Defining the proximal interaction networks of Arf GTPases reveals a mechanism for the regulation of PLD1 and PI4KB (2022)
Figure 7

Fig. 7 ARL5A/5B promote PI4P generation and protein secretion dependent on PI4KB

(A) Schematic representation of the Rapamycin induced mitochondria translocation of ARL5A/5B. The FRB domain was fused with the Tom20-derived mitochondrial anchor sequence. ARL5A/5B were fused with RFP and FKPB. Rapamycin induces the dimer formation of FRB and FKPB, therefore recruiting the FKBP-RFP-ARL5A/5B to mitochondria.

(B) ARL5A/5B recruitment to mitochondria induces PI4P accumulation at the mitochondria. Tom20-CFP-RFB and P4Mx2-mEGFP were co-expressed with RFP-FKBP vector or RFP-FKBP-ARL5A/5BQ70L constructs in HEK293A cells. Cell samples were treated with or without 100nM rapamycin for 1 hour. Scale bars: 10 μm. The dashed line box indicates the merged signal of PI4P with ARL5A/5B or Tom20. The result is a representative of two biologically independent experiments.

(C) PI4KB knockout blocks the PI4P accumulation induced by mitochondria anchored ARL5A/5B. Tom20-CFP-RFB and P4Mx2-mEGFP were co-expressed with RFP-FKBP vector or RFP-FKBP-ARL5A/5BQ70L constructs in HEK293A PI4KB knockout cells (clone#2-5). Cell samples were treated with or without 100nM rapamycin for 1 hour before imaging. Scale bars: 10 μm. The dashed line box indicates the merged signal of PI4P with ARL5A/5B or Tom20. The result is a representative of two biologically independent experiments.

(D) Mander's overlap coefficient of (B and C, result of rapamycin treatment). Mean± SD; n= 5 technically independent samples. Two-way ANOVA; *P < 0.05.

(E) ARL5A/5B double knockout decreases protein secretion. Gaussia-luciferase was expressed in HEK293A wild-type or three ARL5A/5B double knockout clones as indicated. After 24 hours, the medium was replaced with the fresh medium. After 4 hours, medium was collected, and the secreted Gaussia-luciferase was detected by Western. Cells were also collected and probed with indicated antibodies

(F) ARL5A/5B double knockout decreases protein secretion. Experiments were similar to panel (E). Medium was collected at the indicated time points and measured for luciferase activity. The cellular firefly luciferase activity was measured as the transfection control. Details were described in the method part. 3 ug/ml BFA was used as a positive control to inhibit protein secretion. Data are presented as mean± SD, n = 3 biologically independent samples. One-way ANOVA; *P < 0.05

(G) PI4KB knockout blocks protein secretion stimulated by ARL5A/5BQ70L. Gaussia-luciferase was co-expressed with HA-tagged ARL5AQ70L or ARL5BQ70L in HEK293A wild-type or two PI4KB knockout cell lines as indicated. After 24 hours, the medium was replaced with the fresh medium. After 3 hours, medium was collected and measured for Gaussia-luciferase activity. Data are presented as mean± SD, n = 3 biologically independent samples. Two-way ANOVA, n.s denotes not significant, *P < 0.05.

(H) PI4KB knockout blocks protein secretion stimulated by ARL5A/5BQ70L. Experiments were similar to panel (G). Gaussia-luciferase in culture medium was detected by Western and the cell lysate was immunoblotted with indicated antibodies.

(I) A proposed model of PI4KB regulation by ARL5A/5B. In addition to the reported ARF1, ACBD3, and RAB11A/11B, ARL5A/5B act as novel regulators of PI4KB to promote PI4P synthesis and protein secretion.