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Assembly of the AAA ATPase Vps4 on ESCRT-III.

Shestakova A, Hanono A, Drosner S, Curtiss M, Davies BA, Katzmann DJ, Babst M - Mol. Biol. Cell (2010)

Bottom Line: The order of events leading to active, ESCRT-III-associated Vps4 is poorly understood.Although no single interaction was found to be essential for the localization or activity of Vps4, certain interactions proved more important than others.The most significant among these were the binding of Vps4 to Vta1 and to the ESCRT-III subunits Vps2 and Snf7.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City, UT 84112-9202, USA.

ABSTRACT
Vps4 is a key enzyme that functions in endosomal protein trafficking, cytokinesis, and retroviral budding. Vps4 activity is regulated by its recruitment from the cytoplasm to ESCRT-III, where the protein oligomerizes into an active ATPase. The recruitment and oligomerization steps are mediated by a complex network of at least 12 distinct interactions between Vps4, ESCRT-III, Ist1, Vta1, and Did2. The order of events leading to active, ESCRT-III-associated Vps4 is poorly understood. In this study we present a systematic in vivo analysis of the Vps4 interaction network. The data demonstrated a high degree of redundancy in the network. Although no single interaction was found to be essential for the localization or activity of Vps4, certain interactions proved more important than others. The most significant among these were the binding of Vps4 to Vta1 and to the ESCRT-III subunits Vps2 and Snf7. In our model we propose the formation of a recruitment complex in the cytoplasm that is composed of Did2-Ist1-Vps4, which upon binding to ESCRT-III recruits Vta1. Vta1 in turn is predicted to cause a rearrangement of the Vps4 interactions that initiates the assembly of the active Vps4 oligomer.

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MIM1 and MIM2 interactions contribute to the recruitment of Vps4 to ESCRT-III. (A) Fluorescence microscopy analysis of the Vps4 MIT domain fused to GFP (MIT-GFP). The wild-type, MIM1 mutant (L64D), or MIM2 mutant (I18D) version of MIT-GFP was expressed in different yeast strains (see Table 1), and the extent of endosomal localization was determined (Loc.). For better visualization the fluorescence microscopy pictures were inverted and the intensity was adjusted to the individual brightness range (black is the brightest signal). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure 1A). Numbers in parentheses indicate partially disrupted interactions. (B) Quantification of endosome-localized MIT-GFP relative to wild type (0.0) and vps4Δ (1.0). The data shown represent the results of at least 15 individually analyzed cells. Numbers refer to the experiment number in A. (C) Subcellular fractionation of different yeast strains expressing vps4E233Q into soluble, cytoplasmic fraction (S) and pelletable, membrane-associated fraction (P). Fractions were analyzed by Western blot using antibodies specific for Vps4 (top panels), Snf7 (bottom panel, lanes 9–14) and the HA-tag (bottom panels, lanes 1–8 and 15–16). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure 1A). Numbers in parentheses indicate partially disrupted interactions. (D) In vitro Vps4 interaction studies using wild-type and MIM2 mutant forms of GST-Vps20(C) (fusion of GST with C-terminal half of Vps20) or GST-Snf7(C) immobilized on GSH-Sepharose. Vps4E233Q was added in the presence of ATP or ADP to immobilized proteins; bound and unbound fractions were analyzed by SDS-PAGE and Coomassie staining.
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Figure 2: MIM1 and MIM2 interactions contribute to the recruitment of Vps4 to ESCRT-III. (A) Fluorescence microscopy analysis of the Vps4 MIT domain fused to GFP (MIT-GFP). The wild-type, MIM1 mutant (L64D), or MIM2 mutant (I18D) version of MIT-GFP was expressed in different yeast strains (see Table 1), and the extent of endosomal localization was determined (Loc.). For better visualization the fluorescence microscopy pictures were inverted and the intensity was adjusted to the individual brightness range (black is the brightest signal). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure 1A). Numbers in parentheses indicate partially disrupted interactions. (B) Quantification of endosome-localized MIT-GFP relative to wild type (0.0) and vps4Δ (1.0). The data shown represent the results of at least 15 individually analyzed cells. Numbers refer to the experiment number in A. (C) Subcellular fractionation of different yeast strains expressing vps4E233Q into soluble, cytoplasmic fraction (S) and pelletable, membrane-associated fraction (P). Fractions were analyzed by Western blot using antibodies specific for Vps4 (top panels), Snf7 (bottom panel, lanes 9–14) and the HA-tag (bottom panels, lanes 1–8 and 15–16). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure 1A). Numbers in parentheses indicate partially disrupted interactions. (D) In vitro Vps4 interaction studies using wild-type and MIM2 mutant forms of GST-Vps20(C) (fusion of GST with C-terminal half of Vps20) or GST-Snf7(C) immobilized on GSH-Sepharose. Vps4E233Q was added in the presence of ATP or ADP to immobilized proteins; bound and unbound fractions were analyzed by SDS-PAGE and Coomassie staining.

Mentions: The MIT domain of Vps4 has been shown to interact with six different proteins of the ESCRT machinery: the four subunits of ESCRT-III (Vps2, Vps20, Vps24, Snf7) and two factors that are related to ESCRT-III subunits and have been implicated in the endosomal recruitment of Vps4, Did2, and Ist1 (Figure 1A; Shim et al., 2007; Azmi et al., 2008; Dimaano et al., 2008; Kieffer et al., 2008; Xiao et al., 2008). These six factors bind via two distinct binding motifs, MIM1 and MIM2, to two different surface areas of the MIT domain (Figure 1C). This arrangement allows the MIT domain to simultaneous bind to MIM1 and MIM2. To test which of the MIT interactions are involved in the recruitment of Vps4 to ESCRT-III, we expressed a MIT-GFP fusion construct in yeast strains deleted for VPS4 alone or in combination with other ESCRT mutations. These yeast cells were analyzed by fluorescence microscopy, and the resulting pictures were judged by the ratio of MIT-GFP signal localized either to the cytoplasm or to the aberrant endosomes formed in these mutant strains (class E compartments; Figure 2A). Enlarged examples of the microscopy pictures are shown in Supplemental Figure 1. Furthermore, for a subset of MIT-GFP localization experiments, the ratio of endosomal-to-cytoplasmic localization was quantified (Figure 2B).


Assembly of the AAA ATPase Vps4 on ESCRT-III.

Shestakova A, Hanono A, Drosner S, Curtiss M, Davies BA, Katzmann DJ, Babst M - Mol. Biol. Cell (2010)

MIM1 and MIM2 interactions contribute to the recruitment of Vps4 to ESCRT-III. (A) Fluorescence microscopy analysis of the Vps4 MIT domain fused to GFP (MIT-GFP). The wild-type, MIM1 mutant (L64D), or MIM2 mutant (I18D) version of MIT-GFP was expressed in different yeast strains (see Table 1), and the extent of endosomal localization was determined (Loc.). For better visualization the fluorescence microscopy pictures were inverted and the intensity was adjusted to the individual brightness range (black is the brightest signal). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure 1A). Numbers in parentheses indicate partially disrupted interactions. (B) Quantification of endosome-localized MIT-GFP relative to wild type (0.0) and vps4Δ (1.0). The data shown represent the results of at least 15 individually analyzed cells. Numbers refer to the experiment number in A. (C) Subcellular fractionation of different yeast strains expressing vps4E233Q into soluble, cytoplasmic fraction (S) and pelletable, membrane-associated fraction (P). Fractions were analyzed by Western blot using antibodies specific for Vps4 (top panels), Snf7 (bottom panel, lanes 9–14) and the HA-tag (bottom panels, lanes 1–8 and 15–16). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure 1A). Numbers in parentheses indicate partially disrupted interactions. (D) In vitro Vps4 interaction studies using wild-type and MIM2 mutant forms of GST-Vps20(C) (fusion of GST with C-terminal half of Vps20) or GST-Snf7(C) immobilized on GSH-Sepharose. Vps4E233Q was added in the presence of ATP or ADP to immobilized proteins; bound and unbound fractions were analyzed by SDS-PAGE and Coomassie staining.
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Figure 2: MIM1 and MIM2 interactions contribute to the recruitment of Vps4 to ESCRT-III. (A) Fluorescence microscopy analysis of the Vps4 MIT domain fused to GFP (MIT-GFP). The wild-type, MIM1 mutant (L64D), or MIM2 mutant (I18D) version of MIT-GFP was expressed in different yeast strains (see Table 1), and the extent of endosomal localization was determined (Loc.). For better visualization the fluorescence microscopy pictures were inverted and the intensity was adjusted to the individual brightness range (black is the brightest signal). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure 1A). Numbers in parentheses indicate partially disrupted interactions. (B) Quantification of endosome-localized MIT-GFP relative to wild type (0.0) and vps4Δ (1.0). The data shown represent the results of at least 15 individually analyzed cells. Numbers refer to the experiment number in A. (C) Subcellular fractionation of different yeast strains expressing vps4E233Q into soluble, cytoplasmic fraction (S) and pelletable, membrane-associated fraction (P). Fractions were analyzed by Western blot using antibodies specific for Vps4 (top panels), Snf7 (bottom panel, lanes 9–14) and the HA-tag (bottom panels, lanes 1–8 and 15–16). Vps4 interactions affected by the different mutations are listed (Int., numbers are based on the interactions in Figure 1A). Numbers in parentheses indicate partially disrupted interactions. (D) In vitro Vps4 interaction studies using wild-type and MIM2 mutant forms of GST-Vps20(C) (fusion of GST with C-terminal half of Vps20) or GST-Snf7(C) immobilized on GSH-Sepharose. Vps4E233Q was added in the presence of ATP or ADP to immobilized proteins; bound and unbound fractions were analyzed by SDS-PAGE and Coomassie staining.
Mentions: The MIT domain of Vps4 has been shown to interact with six different proteins of the ESCRT machinery: the four subunits of ESCRT-III (Vps2, Vps20, Vps24, Snf7) and two factors that are related to ESCRT-III subunits and have been implicated in the endosomal recruitment of Vps4, Did2, and Ist1 (Figure 1A; Shim et al., 2007; Azmi et al., 2008; Dimaano et al., 2008; Kieffer et al., 2008; Xiao et al., 2008). These six factors bind via two distinct binding motifs, MIM1 and MIM2, to two different surface areas of the MIT domain (Figure 1C). This arrangement allows the MIT domain to simultaneous bind to MIM1 and MIM2. To test which of the MIT interactions are involved in the recruitment of Vps4 to ESCRT-III, we expressed a MIT-GFP fusion construct in yeast strains deleted for VPS4 alone or in combination with other ESCRT mutations. These yeast cells were analyzed by fluorescence microscopy, and the resulting pictures were judged by the ratio of MIT-GFP signal localized either to the cytoplasm or to the aberrant endosomes formed in these mutant strains (class E compartments; Figure 2A). Enlarged examples of the microscopy pictures are shown in Supplemental Figure 1. Furthermore, for a subset of MIT-GFP localization experiments, the ratio of endosomal-to-cytoplasmic localization was quantified (Figure 2B).

Bottom Line: The order of events leading to active, ESCRT-III-associated Vps4 is poorly understood.Although no single interaction was found to be essential for the localization or activity of Vps4, certain interactions proved more important than others.The most significant among these were the binding of Vps4 to Vta1 and to the ESCRT-III subunits Vps2 and Snf7.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Utah, Salt Lake City, UT 84112-9202, USA.

ABSTRACT
Vps4 is a key enzyme that functions in endosomal protein trafficking, cytokinesis, and retroviral budding. Vps4 activity is regulated by its recruitment from the cytoplasm to ESCRT-III, where the protein oligomerizes into an active ATPase. The recruitment and oligomerization steps are mediated by a complex network of at least 12 distinct interactions between Vps4, ESCRT-III, Ist1, Vta1, and Did2. The order of events leading to active, ESCRT-III-associated Vps4 is poorly understood. In this study we present a systematic in vivo analysis of the Vps4 interaction network. The data demonstrated a high degree of redundancy in the network. Although no single interaction was found to be essential for the localization or activity of Vps4, certain interactions proved more important than others. The most significant among these were the binding of Vps4 to Vta1 and to the ESCRT-III subunits Vps2 and Snf7. In our model we propose the formation of a recruitment complex in the cytoplasm that is composed of Did2-Ist1-Vps4, which upon binding to ESCRT-III recruits Vta1. Vta1 in turn is predicted to cause a rearrangement of the Vps4 interactions that initiates the assembly of the active Vps4 oligomer.

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