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. 2013 Jun 20;8(6):e67307.
doi: 10.1371/journal.pone.0067307. Print 2013.

The N-terminal domains of Vps3 and Vps8 are critical for localization and function of the CORVET tethering complex on endosomes

Nadine Epp  1 , Christian Ungermann
Affiliations

The N-terminal domains of Vps3 and Vps8 are critical for localization and function of the CORVET tethering complex on endosomes

Nadine Epp et al. PLoS One. .

Abstract

Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.

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Conflict of interest statement

Competing Interests: The authors declare that no competing interests exist.

Figures

Figure 1
Figure 1. Consequences of domain deletions on CORVET functionality.
(A) Domain organization of the CORVET-specific subunits Vps8 and Vps3. N-terminal domains (NTDs/ΔCTDs) correspond to putative β-propeller, whereas C-terminal domains (CTDs/ΔNTDs) represent predicted α-solenoids segments. Domain boundaries were determined by the PredictProtein algorithm (www.predictprotein.org). (B) Purification of truncated Vps3 and Vps8 constructs. Small scale tandem affinity purification (TAP) of Vps8 and Vps3 fragments was carried out via IgG Sepharose. Glycin pH 2.5 - eluates were subjected to SDS-PAGE and Western-Blot via the Odyssey scanning system. Immunoprecipitated CORVET subunits were detected by antibodies against Cbp, Vps11 and HA. Expression of truncated constructs was confirmed by decoration eluates of the pull-down with anti-Cbp antibodies (top panel). Sizes are indicated in kDa. (C) Vacuole morphology and endocytic sorting in Vps3 and Vps8 mutants. Sorting of N-terminally GFP-tagged Cps1, expressed from CEN-plasmids, was detected by fluorescence microscopy. To monitor vacuole-morphology, cells were stained with FM4–64. Size bar, 10 µm. See methods for details. (D) Transport of carboxypeptidase Y (CPY). Sorting was monitored by detection of processed CPY (mCPY) from vacuole enriched pellet fractions. Absence of mCPY is due to secretion and defective vacuolar protein sorting. Blots were decorated against Tom40 as loading control.
Figure 2
Figure 2. Localization of CORVET requires the N-terminal domains of Vps3 or Vps8.
(A) Localization of N-terminally truncated Vps3 and Vps8. Vps3 and Vps8 were genomically tagged with 3xmCherry at their C-temini, and colocalized with genomically tagged GFP-Pep12. Size bar, 10 µm. (B) Colocalization of Vps8 with Vps3. Analysis was performed as in A. Vps3 was genomically tagged with 3xmCherry at the C-terminus, whereas Vps8 was tagged with yeGFP. Size bar, 10 µm. (C) Subcellular localization of dually truncated CORVET. TAP-tagged Vps8 was monitored in wild-type and in cells expressing Vps3ΔN and truncated Vps8. Cells (Total, T) were fractionated to obtain a pellet (P100) and supernatant (S100) after the final centrifugation at 100,000 g. Western blots were decorated against the TAP tag to identify Vps8. Decoration with antibodies against Arc1 and Vac8 was used as control for cytosolic and membrane-enriched fractions. (D) Endocytosis of Ste3 in cells expressing truncated Vps3 and Vps8. Endocytosis in the respective strains was followed by fluorescence microscopy of C-terminally GFP-tagged Ste3, expressed from 2µ-plasmids. To monitor vacuole morphology in parallel, cells were treated with FM4–64 beforehand. Size bar, 10 µm.
Figure 3
Figure 3. Assembly of CORVET in the absence of the N-terminal domains of Vps3 and Vps8.
(A,B) Functional rescue of truncated CORVET. (A) CPY secretion. Membrane fractions were generated from indicated strains carrying TAP- and HA-tagged CORVET subunits. Proteins were resolved on SDS-PAGE gels, and Western blots were decorated using anti-CPY and anti-Tom40 antibodies. Arrows in front of ΔN (deletion of the N-terminal domain) indicate overexpression of the respective constructs, whereas+symbols refer to the presence of full-length proteins. (B) Localization of truncated CORVET. Truncated GFP-tagged Vps3 and Vps8, expressed from CEN-plasmids in the respective deletion backgrounds, were followed by fluorescence microscopy in cells where the respective other truncated CORVET subunit was expressed from its endogenous promoter (top) or overexpressed (bottom). (C) Growth sensitivity of truncated CORVET to the toxic arginine homologue canavanine. Strains were grown to logarithmic phase and serial dilutions were spotted onto SDC plates containing indicated concentrations of canavanine . Growth sensitivity is the result of stabilization of the Can1 transporter at the plasma membrane as a consequence of defective endocytosis. (D) Purification of CORVET from mutant cells. Vps8 was C-terminally TAP-tagged and purified using IgG sepharose from wild-type strain and strains expressing truncated versions of CORVET as described in A. Proteins bound to the IgG beads were eluted by TEV protease digestion, and resolved by SDS-PAGE. Western blots were decorated with antibodies against the CbP, HA or Vps11. Where indicated, low exposures are shown. (E) Large scale purification of CORVET and ΔNTD-CORVET. Strains carried TAP-tagged Vps8 and GFP-tagged Vps3. Purification was done as described in Methods. The TEV-eluates were applied to 10%–40% glycerol gradients, and centrifuged at 285,000 g for 18 h at 4°C. Load indicates 20% of the total eluate. TCA-precipitated proteins of collected 1 ml fractions were loaded onto SDS-PAGE and visualized by coomassie-staining. Cbp, calmodulin-binding peptide that remains on protein after TEV cleavage; 3ΔN-GFP, Vps3ΔN-GFP; 8ΔN-CbP, Vps8ΔN-CbP. The slight band in lane 3 of the eluate that shows Vps8 full-length is due to a slight spill-over from lane 2 as all strains express only the indicated CORVET variant. (F) Purification of CORVET from Rab deletion strains. CORVET was purified via Vps8-TAP from either vps21Δ or vps21Δ ypt52Δ ypt53Δ ypt7Δ (rab5/7Δ) strains as described for part D. Blots were decorated against the HA-tag on Vps3 and Vps11.
Figure 4
Figure 4. Connection of N-terminal domains of CORVET-specific subunits to the Rab Vps21.
(A) Localization of ΔNTD-CORVET upon overproduction of Vps21. TEF1pr-VPS21 was introduced via integrative pRS406 based plasmids into the indicated background-strains expressing the fluorescently tagged CORVET versions. Cells were monitored by fluorescence microscopy. Statistical analysis of cells with dot-localized CORVET was done by evaluating 400 cells per indicated strain. Cells with multiple dots (at least two) of colocalized Vps3 and Vps8 were counted and given by percentages. Error-bars represent standard deviation of different frames of 50 cells each. Size bar, 10 µm. (B) Functional analysis of Vps21-overexpression in truncated CORVET strains. CPY secretion assay was performed as before (Figure 1D) by isolating membrane fractions, followed by SDS-PAGE, western blot and decorating them against CPY, and TOM40 as loading-control. Vps21 was overproduced by genomically replacing the endogenous Vps21-promoter with the strong GPD3-promoter, which is even stronger than the TEF promoter used for our localization assays . (C) Interaction of Vps3 with Vps21. 35 µg of yeast-purified (TAP-purification) Vps3 construct was applied to each pull-down reaction of GSH-beads coupled to GST-Vps21 or GST-Ypt7, which were loaded with GTPγs or GDP beforehand. Eluates were loaded onto SDS-PAGE, followed by western blot, which was decorated against Cbp. SDS-sample buffer was added to beads with bound Rabs afterwards, and proteins were analyzed by SDS-PAGE and Coomassie staining. For details see Methods. (D) Interaction of Vps8 with Vps21. Yeast-2-Hybrid analysis of Vps21-interaction with Vps8 constructs as preys was performed as described in Methods. Growth on QDO (quadruple drop out) plates indicates strong interactions, growth on TDO (triple drop out) refers to weaker interactions . (E) Microscopy images of FM4–64 stained cells overproducing the indicated Vps8 constructs. GFP-Vps21 was used as a fluorescent marker of late endosomal accumulations caused by Vps8 overproduction. Size bar, 10 µm.
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This work was supported by the Sonderforschungsbereich (Collaborative Research Center) 944 (project P11) of the Deutsche Forschungsgemeinschaft (German Research Council), and by the Hans-Mühlenhoff foundation (to CU). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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