RILP interacts with HOPS complex via VPS41 subunit to regulate endocytic trafficking - PubMed (original) (raw)

RILP interacts with HOPS complex via VPS41 subunit to regulate endocytic trafficking

Xiaosi Lin et al. Sci Rep. 2014.

Erratum in

Abstract

The HOPS complex serves as a tethering complex with GEF activity for Ypt7p in yeast to regulate late endosomal membrane maturation. While the role of HOPS complex is well established in yeast cells, its functional and mechanistic aspects in mammalian cells are less well defined. In this study, we report that RILP, a downstream effector of Rab7, interacts with HOPS complex and recruits HOPS subunits to the late endosomal compartment. Structurally, the amino-terminal portion of RILP interacts with HOPS complex. Unexpectedly, this interaction is independent of Rab7. VPS41 subunit of HOPS complex was defined to be the major partner for interacting with RILP. The carboxyl-terminal region of VPS41 was mapped to be responsible for the interaction. Functionally, either depletion of VPS41 by shRNA or overexpression of VPS41 C-terminal half retarded EGF-induced degradation of EGFR. These results suggest that interaction of RILP with HOPS complex via VPS41 plays a role in endocytic trafficking of EGFR.

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Figures

Figure 1

Figure 1. RILP recruits HOPS subunits to endosomal vesicles.

Hela cells were co-transfected with GFP-RILP and myc-tagged human VPS11, VPS16, VPS18, VPS33, VPS39 and VPS41, respectively. VPS proteins are immuno-labeled by 9E10 antibody followed by Texas-red conjugated secondary antibody and viewed along with GFP signal. Immunofuorescence microscopy revealed that RILP recruits all HOPS subunits (except for VPS33) to endosomal vesicles. Bar = 20 μm.

Figure 2

Figure 2. RILP interacts with HOPS complex.

(A). Hela cell lysates derived from cells expressing myc-tagged HOPS subunits, VPS11, VPS16, VPS18, VPS33, VPS39 and VPS41, respectively, were subjected to GST-pulldown assay using immobilized GST-RILP. 9E10 antibody was used for western-blotting to detect the protein bound to GST-RILP. The results revealed that RILP binds to all HOPS subunits except for VPS33, suggesting RILP interacts with HOPS complex. (B). GST-pulldown assay using GST-RILP, GST-RILP (1–198) and GST-RILP(199–401) demonstrated that N-terminal region (1–198aa) of RILP interacts with subunits of HOPS complex. Un-cropped blots/gels are presented in Supplementary Figure s7.

Figure 3

Figure 3. The interaction between RILP and HOPS complex is independent of Rab7.

(A). Hela cells were transfected with pSuper.GFP-scramble-shRNA(lane 1) or shRNA-Rab7. 48 h later, knocked-down cells were transfected with myc-tagged VPS11 (lane 2), VPS16 (lane 3), VPS18 (lane 4), VPS39 (lane 5) and VPS41 (lane 6), respectively. 72 h later, cells were harvested and processed for detection of the knockdown efficiency. The results revealed that Rab7 was depleted efficiently in the cells expressing HOPS subunits. (B). Lysates derived from cells described above were subjected for GST-pulldown assay using GST-RILP, the results demonstrated that RILP can still bind to HOPS subunits when Rab7 was effectively depleted, suggesting RILP interacts with HOPS complex independent of Rab7. (C). Hela lysates containing myc-tagged VPS11, VPS16, VPS18, VPS39 and VPS41 were subjected for GST-pulldown assay using GST-RILP and GST-RILP304AAA306 mutant (defective in interacting with Rab7), the data showed that this RILP mutant can still bind to HOPS sununits, although no longer interacting with Rab7 (bottom panel). (D). Hela lysates containing myc-tagged VPS11, VPS16, VPS18, VPS33, VPS39 and VPS41 were subjected for GST-pulldown assay using GST-Rab7 or GST-Rab7L8A mutant (defective in interacting with RILP showed in bottom panel). Like wildtype Rab7, Rab7L8A can still weakly interacts with HOPS complex, suggesting Rab7 interacts with HOPS complex independent of RILP. Un-cropped blots/gels are presented in Supplementary Figure s8.

Figure 4

Figure 4. VPS41 subunit plays a crucial role in RILP-HOPS interaction.

(A). Hela cells expressing GFP-RILP were immuno-labeled with anti-VPS41 antibody to demonstrate the recruitment of endogenous VPS41 by RILP. (B). Hela cells were co-transfected with pSuper.GFP-shRNA-VPS41, myc-tagged HOPS subunits and HA-RILP, then processed for immuno-staining with myc and HA tag antibodies. Knockdown cells were revealed by co-expresed GFP signal. Myc-tagged proteins were revealed by Texas-red conjugated secondary antibodies and HA-tagged proteins were revealed by Cy5-conjugated secondary antibodies. Immuofluorescence microscopy showed that depletion of VPS41 disrupts the co-localization of other HOPS subunits with RILP. (C). GST-pulldown experiments demonstrated that depletion of VPS41 decreases the amount of HOPS subunits bound to RILP. (D). Quantitative analysis of the results from triplicate experiments revealed that the amount of HOPS subunits bound to RILP is significantly decreased when VPS41 was knocked-down. Un-cropped blots/gels are presented in Supplementary Figure s9. bar = 20 μm.

Figure 5

Figure 5. C-terminal region of VPS41 contributes to the interaction between RILP and HOPS complex.

(A). Purified His-VPS41 or His-Vps33 recombinant protein was incubated with immobilized GST-RILP to show RILP directly interacts with VPS41, but not VPS33. (B). Hela cells were co-expressed with GFP-RILP and myc-VPS41, myc-VPS41(1–427) or VPS41(428–855), respectively. Immunofluorescence microscopy revealed that RILP can recruit wild type VPS41 and VPS41(428–855), but not VPS41(1–427). (C). Pulldown experiments showed that RILP specifically binds to VPS41 through its C-terminal region(428–855aa). Un-cropped blots/gels are presented in Supplementary Figure s10. Bar = 20 μm.

Figure 6

Figure 6. Disruption of HOPS complex inhibits EGFR degradation.

(A). MCF7 cells were transfected with pSuper.GFP-scramble-shRNA or shRNA-VPS41, 48 h later, the cells were starved for overnight, then stimulated with EGF for the indicated times. The protein level of EGFR was examined by western-blot using mAb against EGFR. The results demonstrated EGFR decreased quickly in scramble knocked-down cells, but the degradation of EGFR is compromised in shRNA-VPS41 knocked-down cells. Un-cropped blots/gels are presented in Supplementary Figure s11. (B). Quantitative analysis from 3 independent experiments reveals that EGFR degradation is significantly arrrested in shRNA-VPS41 knocked-down cells. (C). Immunofluorescence microscopy showed rapid endocytic clearance of endocytosed EGF-Rhodamine in scramble knockdown cells. (D). depletion of VPS41 delayed clearance of endocytosed EGF-Rhodamine as evidenced by the accumulation of endocytosed EGF-Rhodamine in structures partially overlapping with EEA1-labeled early endosome after 60 min internalization. (E). EGF-Rhodamine accumulated vesicles were co-localized with late endosomal/lysosomal markers CD63 and Lamp1 after 60 min of endocytosis in cells with VPS41 depletion. Bar = 20 μm.

Figure 7

Figure 7. The effects of overexpressing VPS41(428–855) (VPS41CT) on EGF-Rhodamine endocytosis and degradation.

(A). over-expressing VPS41CT delayed endocytic clearance of EGF-Rhodamine similar to the effect of shRNA-VPS41. (B). EGF-Rhodamine accumulated vesicles were co-localized with late endosomal/lysosomal markers CD63 and Lamp1 after 60 min endocytosis in cells expressing VPS41CT. Bar = 20 μm.

Figure 8

Figure 8. A working model of HOPS-Rab7-RILP interaction on the endosomal membrane.

In this model, Rab7 interacts with both accessory subunits VPS39 and VPS41 of HOPS complex, while RILP interacts with Rab7 and VPS41 to stabilze Rab7-HOPS at the endosomal membrane. (B). a working model of Rab7 modulating endosomal membrane transition regulated by Mon1/Ccz1, HOPS and RILP. In this model, Mon1-Ccz1 plays cis-regulation to exclude Rab5 and recruit HOPS complex, activating Rab7, while RILP plays trans-regulation to stabilize Rab7-HOPS complex onto late endosomal membrane.

References

    1. Bucci C., Thomsen P., Nicoziani P., McCarthy J. & van Deurs B. Rab7: a key to lysosome biogenesis. Mol. Biol. Cell 11, 467–480 (2000). - PMC - PubMed
    1. Ceresa B. P. & Bahr S. J. Rab7 activity affects epidermal growth factor:epidermal growth factor receptor degradation by regulating endocytic trafficking from the late endosome. J.Biol.Chem. 281, 1099–1106 (2006). - PubMed
    1. Nickerson D. P., Brett C. L. & Merz A. J. Vps-C complexes: gatekeepers of endolysosomal traffic. Curr.Opin.Cell.Biol. 21, 543–551 (2009). - PMC - PubMed
    1. Wang T., Zhang M., Wu X. & Hong W. Rab7: Role of its protein interaction cascades in endo-lysosomal traffic. Cell.Signal. 23, 516–521 (2011). - PubMed
    1. Cantalupo G., Alifano P., Roberti V., Bruni C. B. & Bucci C. Rab-interacting lysosomal protein (RILP): the Rab7 effector required for transport to lysosomes. EMBO J. 20, 683–693 (2001). - PMC - PubMed

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