New component of ESCRT-I regulates endosomal sorting complex assembly

Abstract

The endosomal sorting complex required for transport (ESCRT) complexes play a critical role in receptor down-regulation and retroviral budding. Although the crystal structures of two ESCRT complexes have been determined, the molecular mechanisms underlying the assembly and regulation of the ESCRT machinery are still poorly understood. We identify a new component of the ESCRT-I complex, multivesicular body sorting factor of 12 kD (Mvb12), and demonstrate that Mvb12 binds to the coiled-coil domain of the ESCRT-I subunit vacuolar protein sorting 23 (Vps23). We show that ESCRT-I adopts an oligomeric state in the cytosol, the formation of which requires the coiled-coil domain of Vps23, as well as Mvb12. Loss of Mvb12 results in the disassembly of the ESCRT-I oligomer and the formation of a stable complex of ESCRT-I and -II in the cytosol. We propose that Mvb12 stabilizes ESCRT-I in an oligomeric, inactive state in the cytosol to ensure that the ordered recruitment and assembly of ESCRT-I and -II is spatially and temporally restricted to the surface of the endosome after activation of the MVB sorting reaction.

Figure 1.

Mvb12 is a novel protein in the ESCRT pathway. (A) Sorting of GFP-CPS and Ste2-GFP in WT (SEY6210) and mvb12Δ (TCY246). (B) Yeast strain expressing Mvb12-GFP was transformed with dsRed-FYVE, and partial colocalization is observed. (C) Localization of Mvb12-GFP in WT, vps23Δ, vps36Δ, snf7Δ, and vps4Δ strains. Mvb12-GFP endosomal localization depends on Vps23 (ESCRT-I), but not on Vps36 (ESCRT-II), Snf7 (ESCRT-III), or Vps4.

Figure 2.

Mvb12 is a new component of the ESCRT-I complex. (A) Gel filtration analysis of cell extracts from yeast strain expressing Mvb12-Flag (TCY257). Column fractions were analyzed by Western blotting using antibodies specific for Vps23 or Flag. Mvb12-Flag cofractionates with Vps23. (B) Cell extracts from WT (SEY6210) or strain expressing Mvb12-Flag (TCY257) were subjected to immunoprecipitation using antibody specific for Flag. Immunoprecipitates were analyzed by Western blotting using antibodies against Vps23 or Flag. Mvb12-Flag coimmunoprecipitates with Vps23. The asterisk highlights a nonspecific band.

Figure 3.

Mvb12 negatively regulates the interaction between ESCRT-I and -II. (A) Gel filtration analysis of cell extracts from WT (SEY6210) strain expressing Vps36-HA, mvb12Δ strain expressing Vps36-HA, and mvb12Δ vps36Δ strain expressing Vps37-HA. Column fractions were analyzed by Western blotting using antibodies specific for Vps23 or HA. Vps36-HA does not cofractionate with Vps23 in WT, but cofractionates with Vps23 in mvb12Δ as a ∼250-kD complex in the cytoplasm. Deletion of VPS36 from the mvb12Δ strain disrupts the ESCRT-I–II heterodimer to form a ∼100-kD ESCRT-I monomer. (B) Cell extract from mvb12Δ strain expressing control plasmid or Vps36-HA plasmid were immunoprecipitated using antibody specific for HA. Immunoprecipitates were analyzed by Western blotting using antibodies against Vps23 or HA. Vps23 coimmunoprecipitates with Vps36-HA.

Figure 4.

Yeast native ESCRT-I assembles into an oligomer in an Mvb12-dependent manner. (A) Cytosolic ∼350-kD ESCRT-I complex is an oligomer. Cell extracts from Vps23-Myc strain (SJY027) expressing either empty vector or Vps23-Flag (pSJ106); mvb12Δ,Vps23-Myc strain (TCY274) expressing Vps23-Flag (pSJ106) were immunoprecipitated using antibody specific for Flag. Immunoprecipitates were analyzed by Western blotting using antibodies against Myc. Vps23-Flag coimmunoprecipitates with Vps23-Myc in wild-type cells, but not in mvb12Δ cells. The nonspecific band noted by the asterisk in the bottom gel likely corresponds to cross-reactivity of antibody heavy chains (last three lanes of the gel) that were loaded on the gel (anti-FLAG immunoprecipitations) with the goat anti–mouse antibodies used for this Western blot (in lane 5, a minor degradation product of the Vps23-FLAG also was detected at this position). (B; left) Cell extracts from WT cells (control) or cells expressing Myc-tagged Vps23 were prepared, analyzed by BN-PAGE, and immunoblotted using antibodies specific for the Myc tag. (right) Purified E. coli ESCRT-I was analyzed by BN-PAGE (left lane) or treated with 1 mM BS³ before BN-PAGE analysis. The monomeric form of E. coli ESCRT-I, as well as the dimeric and trimeric forms generated upon cross-linking, are labeled and indicated by the arrowheads.

Figure 5.

The coiled-coil domain of Vps23 mediates oligomerization of ESCRT-I. (A) Domain structure of Vps23 and coiled-coil domain mutants used in this study. (B) Mutations in the coiled-coil domain disrupt ESCRT-I complex assembly in vivo as determined by gel filtration analysis. Cell extracts were isolated from WT (SEY6210) and vps23Δ strain expressing Vps23^M283D/L286D and Vps37-HA. Column fractions were analyzed by Western blotting using antibodies specific for Vps23 or HA. Both Vps23^M283D,L286D and Vps37 were detected in a ∼250-kD subcomplex in the cytoplasm of vps23Δ strain similar to that of mvb12Δ strain. (C) Coiled-coil domain mutants displayed a MVB-sorting defect. MVB cargo is visualized using a GFP-CPS fusion, and the limiting membrane of the vacuole is visualized with FM4-64. Shown are Nomarski optics (bottom) and fluorescence localization of GFP-CPS (top) and FM4-64 (middle) in vps23Δ strain expressing WT Vps23, Vps23^M283D,L286D, Vps23^Δ257-299, or Vps23^Δ206-256.

Figure 6.

Mvb12 interacts with the coiled-coil domain of Vps23. (A) Cell extract from strains coexpressing Mvb12-Flag and either wild-type Vps23 or Vps23 with coiled-coil mutations were immunoprecipitated using antibody specific for Flag. Immunoprecipitates were analyzed by Western blot using antibodies against Vps23 or Flag. Mvb12 fails to interact with Vps23 coiled-coil deletion and point mutants. (B) Direct Mvb12 and Vps23 interaction as determined by in vitro pull-down assay. Mvb12-Flag protein were incubated with equal amount of E. coli–expressed Vps23-His₆ and Vps23(Δ206-299)-His₆ proteins preconjugated with Ni²⁺ NTA beads. Bound proteins were eluted and analyzed by Western blot using antibodies against Flag. Mvb12 failed to interact with Vps23 coiled-coil deletion mutant.

Figure 7.

Working model for the function of Mvb12. In the cytosol, Mvb12 associates with and stabilizes ESCRT-I in an oligomeric, inactive state so that ESCRT-I cannot interact with ESCRT-II. Once recruited to the endosome, ESCRT-I assembles with ESCRT-II, leading to the activation of the downstream ESCRT machinery and cargo sorting into the MVB pathway. In the absence of Mvb12, the assembly of ESCRT-I and -II occurs prematurely in the cytosol, resulting in a defect in MVB sorting.