Efficient cargo sorting by ESCRT-I and the subsequent release of ESCRT-I from multivesicular bodies requires the subunit Mvb12 - PubMed (original) (raw)

Efficient cargo sorting by ESCRT-I and the subsequent release of ESCRT-I from multivesicular bodies requires the subunit Mvb12

Matt Curtiss et al. Mol Biol Cell. 2007 Feb.

Abstract

The endosomal sorting complex required for transport (ESCRT)-I protein complex functions in recognition and sorting of ubiquitinated transmembrane proteins into multivesicular body (MVB) vesicles. It has been shown that ESCRT-I contains the vacuolar protein sorting (Vps) proteins Vps23, Vps28, and Vps37. We identified an additional subunit of yeast ESCRT-I called Mvb12, which seems to associate with ESCRT-I by binding to Vps37. Transient recruitment of ESCRT-I to MVBs results in the rapid degradation of Mvb12. In contrast to mutations in other ESCRT-I subunits, which result in strong defects in MVB cargo sorting, deletion of MVB12 resulted in only a partial sorting phenotype. This trafficking defect was fully suppressed by overexpression of the ESCRT-II complex. Mutations in MVB12 did not affect recruitment of ESCRT-I to MVBs, but they did result in delivery of ESCRT-I to the vacuolar lumen via the MVB pathway. Together, these observations suggest that Mvb12 may function in regulating the interactions of ESCRT-I with cargo and other proteins of the ESCRT machinery to efficiently coordinate cargo sorting and release of ESCRT-I from the MVB.

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Figures

Figure 1.

Localization of Mvb12 to MVBs is dependent on Vps27 and ESCRT-I. (A) Fluorescent microscopy of yeast strains (wild type [WT], MBY66; _vps4_Δ, MBY64; _vps4_Δ/_vps27_Δ, MBY22; and _vps4_Δ/_vps37_Δ, MCY19) expressing MVB12-GFP (pMB239). White arrows indicate class E compartments, aberrant endosomal structures present in class E vps mutants. (B) Immunofluorescence microscopy of _vps4_Δ (MBY3) and _vps4_Δ_vps27_Δ (MBY22) expressing MVB12-GFP (pMB239) demonstrates that colocalization of Mvb12 with endosome-associated ESCRT-III subunit Snf7 is dependent on Vps27. The Snf7 protein was visualized using specific anti-Snf7 antibody and fluorescently labeled secondary antibody. (C) Fluorescence microscopy of WT and _vps4_Δ (MBY3) strains expressing VPS23-RFP (pMB320) and MVB12-GFP (pMB321) demonstrates colocalization of Mvb12 with ESCRT-I.

Figure 2.

Mvb12 is unstable in ESCRT-I mutants and physically interacts with the ESCRT-I complex. (A) Western blot analysis using anti-HA antibodies of total cell extracts from yeast strains expressing MVB12-HA (pMB240) or as a negative control (con.) MVB12 (pMB238). The strains used are described in Table 1. (B) Subcellular fractionation of yeast strains expressing MVB12-HA either from a low copy (pMB240) or a high copy (2μ, pCJ2) plasmid. The resulting supernatant (S) and pellet (P) fractions were analyzed by Western blot for the presence of Mvb12-HA and Vps23. (C) Extracts from wild-type cells expressing MVB12 (negative control) or MVB12-HA were used for an immunoprecipitation experiment using anti-HA antibodies. Samples of the immunoprecipitated material (bound, lanes 1 and 2) and the remaining supernatants (lanes 5 and 6) were analyzed by Western blot for the presence of Mvb12-HA and Vps23. Extracts from wild-type yeast expressing MVB12-HA in addition to VPS23 (negative control) or VPS23-ProtA were subjected to affinity purification by using IgG-Sepharose. The resulting enriched material (lanes 3 and 4) and the remaining supernatants (lanes 7 and 8) were analyzed by Western blot for the presence of Mvb12-HA and Vps23.

Figure 3.

Gel filtration experiments suggest that Mvb12 associates with ESCRT-I via the subunit Vps37. (A) Cytosol from different yeast strains expressing either MVB12-HA (pMB240) or HA-MVB12 (pMB301) was separated by gel filtration (Sephacryl S300), and the resulting fractions were analyzed by Western blot for the presence of Mvb12-HA, HA-Mvb12, and Vps23. Gels 8 and 9 show the results of the gel filtration analysis of wild type expressing MVB12-HA after 2 h of cycloheximide treatment. (B) Detailed analysis of gel filtration experiments from Figure 3A. Bars above the gels indicate the pooled fractions used in Figure 3A gels 2, 3, and 8. (C) Total cell extracts were prepared from yeast expressing MVB12-HA at different times after treatment with cycloheximide (0, 1, and 2 h) and analyzed by Western blot using anti-HA and anti-Vps23 antibodies. After 2 h of cycloheximide treatment the cells were prepared for gel filtration analysis and the extract loaded onto the column (L) was analyzed by Western blot. The result of this gel filtration analysis is shown in A (gels 8 and 9). (D) The table summarizes the apparent molecular masses of the analyzed proteins relative to the standard proteins thyroglobulin (670 kDa), ferritin (440 kDa), aldolase (158 kDa), albumin (67 kDa), ovalbumin (44 kDa) and myoglobin (17 kDa) (see A).

Figure 4.

Mvb12 requires Vps23 and Vps37 to localize to endosomes. Fluorescent microscopy of Mvb12-GFP overexpressed from a high-copy plasmid in different mutant backgrounds (arrows indicate class E compartments).

Figure 5.

Rapid turnover of Mvb12 by the proteasome depends on a functional MVB pathway. The translation inhibitor cycloheximide (50 mg/l) was added to yeast cultures (t = 0), and samples were taken after addition of the drug at the indicated time points. Cells were lysed using glass beads, and the resulting extracts were analyzed by Western blot for the presence of Vps23, Mvb12-HA, and Vps37-HA.

Figure 6.

The deletion of MVB12 results in a mild MVB trafficking phenotype. (A) Strains expressing either GFP-CPS (pGO45) or Ste2-GFP (pCS24) were analyzed by fluorescence microscopy. White arrows indicate class E compartments. ESCRT-II was overexpressed by transforming the cells with the high-copy plasmid pMB175 (2μ ESCRT-II). (B) Invertase plate assay of wild type (BHY10), _vps23_Δ (EEY5-2), and _mvb12_Δ (MCY9).

Figure 7.

In _mvb12_Δ, ESCRT-I is mislocalized to the vacuolar lumen. (A) Fluorescence microscopy of strains expressing plasmid encoded GFP-VPS27 (pEE27-4) and VPS23(M85T)-GFP (pMB319) or chromosomally integrated VPS23-GFP and VPS36-GFP. Arrows indicate class E compartments. (B) Western blot analysis using a GFP-specific antibody of extracts from cells expressing chromosomally encoded Vps23-GFP (WT, DKY54; _mvb12_Δ, MBY73; 2μ ESCRT-II, pMB175).

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Efficient cargo sorting by ESCRT-I and the subsequent release of ESCRT-I from multivesicular bodies requires the subunit Mvb12 - PubMed (original) (raw)