The CORVET complex promotes tethering and fusion of Rab5/Vps21-positive membranes - PubMed (original) (raw)
The CORVET complex promotes tethering and fusion of Rab5/Vps21-positive membranes
Henning J kleine Balderhaar et al. Proc Natl Acad Sci U S A. 2013.
Abstract
Membrane fusion along the endocytic pathway occurs in a sequence of tethering, docking, and fusion. At endosomes and vacuoles, the CORVET (class C core vacuole/endosome tethering) and HOPS (homotypic fusion and vacuole protein sorting) tethering complexes require their organelle-specific Rabs for localization and function. Until now, despite the absence of experimental evidence, it has been assumed that CORVET is a membrane-tethering factor. To test this theory and understand the mechanistic analogies with the HOPS complex, we set up an in vitro system, and establish CORVET as a bona-fide tether for Vps21-positive endosome/vacuole membranes. Purified CORVET binds to SNAREs and Rab5/Vps21-GTP. We then demonstrate that purified CORVET can specifically tether Vps21-positive membranes. Tethering via CORVET is dose-dependent, stimulated by the GEF Vps9, and inhibited by Msb3, the Vps21-GAP. Moreover, CORVET supports fusion of isolated membranes containing Vps21. In agreement with its role as a tether, overexpressed CORVET drives Vps21, but not the HOPS-specific Ypt7 into contact sites between vacuoles, which likely represent vacuole-associated endosomes. We therefore conclude that CORVET is a tethering complex that promotes fusion of Rab5-positive membranes and thus facilitates receptor down-regulation and recycling at the late endosome.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Purification and characterization of CORVET. (A) Construction of CORVET overexpression strains. Overproduction strains with a C-terminal TAP-tag on the CORVET specific subunits were created by mating of haploid strains overexpressing the class C core proteins (MAT_alpha_) or tagged CORVET specific subunits (MAT_a_), respectively. (B) Tandem affinity purification of CORVET and HOPS. Subunits of the Vps tethers were co-overexpressed and purified from yeast lysate with the TAP method, using Vps3, Vps8 (CORVET), and Vps41 (HOPS) as bait proteins (for details see Materials and Methods). Next, 2.5% of the eluates were mixed with SDS sample buffer, resolved on gradient 7.5% SDS/PAGE gels, and stained with Coomassie. Asterisks (*) indicate subunits with remaining calmodulin-binding peptide (CbP). (C) Purification of CORVET via a glycerol gradient. The CORVET complex was isolated via TAP-tagged Vps8 (as described in B), and the TEV-eluate was applied on a 10–40% (wt/vol) gradient. Fractions were harvested, TCA precipitated, and analyzed by SDS-PAGE and Coomassie staining. A quantification of the protein amount is shown above the SDS-PAGE. (D) Purified CORVET interacts with Vps21-GTP. GST-Rab fusion proteins of Vps21, Ypt7, and Ypt1 were loaded with the respective nucleotides, bound to GSH beads, and used for the Rab pull-down experiment. CORVET was purified from overexpression strain via the TAP-tagged Vps8 subunit (as shown in B and C) and added to immobilized Rabs. After a 1.5-h incubation, eluted proteins and GST-Rabs were analyzed by SDS/PAGE and Western blotting against the CbP-tag (Vps8) and Vps18. (E) Interaction of CORVET with the endosomal SNARE Pep12. Purified CORVET (B–D) was added to immobilzed (SNARE-) GST constructs. Samples were incubated for 2 h, then washed and eluted by boiling in SDS sample buffer. Proteins were analyzed as described in D. (F) CORVET interacts with assembled vacuolar SNAREs. GST-Vam7 SNARE domain (GST-V7-SD) or GST were incubated with His-Vti1 and His-Vam3∆N (His-V3∆N) in the presence of CORVET or HOPS for 2 h. After coupling to GSH beads and washing, the assembly of the SNAREs was confirmed by SDS-PAGE and Coomassie staining (Lower), whereas CORVET (and HOPS) binding was shown by Western blotting (Upper) and antibody decoration against the Calmodulin peptide (on Vps8 in CORVET and on Vps41 in HOPS).
Fig. 2.
Tethering of endolysosomal membranes by the CORVET complex. (A) Purified HOPS and CORVET variants. Overexpressed CORVET and HOPS with or without GFP-tags on the indicated subunits were purified via TAP-tags on Vps8 (CORVET) or Vps41 (HOPS), as described in Materials and Methods, separated on SDS-PAGE gel, and stained with Coomassie. The lane between 4 and 5 was not loaded with protein. (B) Tethering controls. Isolated vacuoles were visualized directly by differential interference contrast (DIC) optics (mock) or incubated with Ficoll buffer (25% of volume, 10 mM Pipes/KOH, pH 6.8, 200 mM sorbitol), TAP buffer (25% of volume), Mon1-Ccz1 [1.1 µM, purified via Ccz1 (9)], BSA (2.4 µM), or GST (40.0 µM). (C–F) Induction of tethering by HOPS and CORVET. Tethering was monitored via microscopy by DIC optics or by following GFP-fluorescence. Purified CORVET (200 nM) and HOPS (800 nM) had a C-terminal GFP-tag on Vps33 (C and D) or Vps3 (E). (F) CORVET without a GFP-tag was added. (G) Quantification of tethering mediated by CORVET-GFP. CORVET with Vps33-GFP was added at the indicated concentrations, and tethered vacuoles were counted. To quantify the results, vacuoles were counted for each experimental condition (n > 900) and mean values ± SDs were calculated. For details see Materials and Methods. (H–K) Localization of Rabs and endosomal proteins that are associated with purified vacuoles. Vacuoles were purified from cells expressing RFP-tagged Vps21 (H), Vps3 (J), or Ypt7 (I), or GFP-tagged Pep12 (K), and visualized by DIC optics and fluorescence microcopy. (L) Colocalization of CORVET and Vps21. RFP-Vps21-positive vacuoles were incubated with CORVET-GFP (Vps33). Dot-like structures observed by DIC optics (Upper) were monitored by fluorescence microscopy (enlarged structures, Lower). (M) Titration of CORVET. CORVET-GFP (Vps33) was added at the indicated concentrations to the tethering assay. All reactions contained the same salt concentration (110 mM NaCl). Vacuoles were monitored as before. Yellow squares mark enlarged images. (Scale bars: 5 µm in B–F; 2 µm in H–K and M, 3 µm in L)
Fig. 3.
CORVET promotes tethering and fusion in a Rab-dependent manner. (A and B) Effect of Msb3 on CORVET-mediated fusion. (A) Tethering in the presence and absence of Msb3. Vacuoles/endosomes isolated from wild-type (wt) or _msb3_∆ cells were preincubated for 5 min with Ficoll buffer or, where indicated, with Msb3 (140 nM). After addition of CORVET-GFP (Vps33, 135 nM), vacuoles were analyzed via microscopy. (Scale bar, 5 µm.) (B) CORVET-GFP titration and quantification of tethered vacuolar structures. CORVET was added as described in A, in total n = 12,800 vacuoles were analyzed (> 500 vacuoles at each concentration), mean values and ± SEM were calculated. (C) Effect of Vps9 on CORVET-mediated tethering. Vacuoles were purified from wild-type or cells expressing Vps21 Q66L, and subjected to the tethering assay in the presence of 125 nm CORVET. Where indicated, recombinant purified Vps9 (1.5 µM) or Msb3 (140 nM) were added. All reactions were supplied with 0.1 mM GTP. Quantification was done as in B. (D) Fusion of vacuoles/endosomes from _msb3_∆ cells in the presence of Vam7. Vacuoles/endosomes from the two tester strains were isolated and incubated in the presence of indicated amounts of recombinant Vam7. Fusion was determined after 90 min at 26 °C (Materials and Methods). (Inset) Titration of Vam7 was plotted against a logarithmic scale of protein concentration. (E) CORVET stimulates the Vam7-dependent fusion reaction in vitro. The same vacuoles/endosomes as in D were incubated with 6.8 nM Vam7 in the presence of indicated amounts of the purified CORVET complex, HOPS complex, or buffer as a control. The extent of fusion was determined after 90 min at 26 °C. For every titration curve, three different experiments were carried out. Mean values with SDs were plotted against protein concentrations after subtraction of respective buffer values. D shows a representative experiment (n = 3).
Fig. 4.
Relocalization of Vps21 upon CORVET overproduction. (A and B) Accumulation of overproduced CORVET at vacuole-vacuole contact sites. Cells expressing all CORVET subunits under control of the GAL1 promoter were grown in YPG medium to midlogarithmic phase, stained with FM4-64, harvested, washed, and analyzed by fluorescence microscopy. The GFP-tag was fused C-terminally to the indicated overproduced subunits. (B) Enlarged image section of Vps11-GFP in the CORVET overexpression strain (A). (C) Localization of RFP-tagged Rab proteins Vps21 and Ypt7 in wild-type (wt) cells. Cells were grown in (SRC-Met + 2% gal) for plasmid maintenance and analyzed as described. Exposure times were kept constant for all strains and approaches in A–C. (D and E) Colocalization of RFP-Rabs and overproduced CORVET-GFP. Cells were incubated in the same selective medium as described in C to induce overexpression of CORVET and maintenance of RFP-Rab encoding plasmids. The GFP-tag was fused to the CORVET subunit Vps33. Exposure times were kept constant for all strains and approaches. (A–E) Depicted fluorescence images are sum projections of four _z_-slices (0.25-µm spacing), after 3D deconvolution. (F and G) Clustering of endolysosomal structures upon CORVET overexpression. YPT7 was deleted in wt (F) and CORVET overproduction (G) strains that lack an additional GFP-tag. Cells were grown in YPD to repress, or YPG to induce the GAL1 promoter-driven overexpression of CORVET, and analyzed as described in A. (Scale bars, 5 µm in A, F, and G; 3 µm in B–E.)
References
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