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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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Localization of mutant Vps4 proteins. (A) Localization of MIT-deleted Vps4 (GFP- Vps4ΔMIT,E233Q and GFP-Vps4ΔMIT,E233Q,S377A) in different yeast mutant strains (see Table 1) determined by fluorescence microscopy (FM). (A and D) The extent of observed endosomal localization is indicated (Loc.). Vps4 interactions affected by the different mutations are listed. Int., numbers are based on the interactions in Figure 1A. (B) Endosomal recruitment of MIT-deleted Vps4 in the presence or absence of full-length Vps4 protein determined by subcellular fractionation and Western blot analysis (S, soluble; P, pellet). (C) Immunoprecipitation of Vps20-HA from detergent-solubilized membrane fractions. The resulting bound and unbound samples were analyzed by Western blot using anti-Vps20 antiserum. (D) Fluorescence microscopy (FM) analysis of GFP-tagged full-length Vps4 protein in different mutant strains (Table 1). Numbers in parentheses indicate partially disrupted interactions.
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Figure 4: Localization of mutant Vps4 proteins. (A) Localization of MIT-deleted Vps4 (GFP- Vps4ΔMIT,E233Q and GFP-Vps4ΔMIT,E233Q,S377A) in different yeast mutant strains (see Table 1) determined by fluorescence microscopy (FM). (A and D) The extent of observed endosomal localization is indicated (Loc.). Vps4 interactions affected by the different mutations are listed. Int., numbers are based on the interactions in Figure 1A. (B) Endosomal recruitment of MIT-deleted Vps4 in the presence or absence of full-length Vps4 protein determined by subcellular fractionation and Western blot analysis (S, soluble; P, pellet). (C) Immunoprecipitation of Vps20-HA from detergent-solubilized membrane fractions. The resulting bound and unbound samples were analyzed by Western blot using anti-Vps20 antiserum. (D) Fluorescence microscopy (FM) analysis of GFP-tagged full-length Vps4 protein in different mutant strains (Table 1). Numbers in parentheses indicate partially disrupted interactions.

Mentions: The Vps4 L64D mutation caused a severe GFP-CPS sorting defect indicating that the loss of all MIM1 interactions blocked Vps4 function (Figure 3A, 4). In contrast, the MIM2-interaction mutant Vps4I18D only partially inhibited the activity of Vps4 (Figure 3A, 3), which is consistent with the observed partial loss of MITI18D-GFP recruitment to endosomes (Figure 2, A and B, 10). However, single and double mutants of the ESCRT-III MIM2 motifs resulted in normal GFP-CPS trafficking, further supporting the notion that mutants with weak MIT-localization defects show correspondingly weak (or no) MVB-trafficking phenotypes (Figure 3A, 14–16).


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)

Localization of mutant Vps4 proteins. (A) Localization of MIT-deleted Vps4 (GFP- Vps4ΔMIT,E233Q and GFP-Vps4ΔMIT,E233Q,S377A) in different yeast mutant strains (see Table 1) determined by fluorescence microscopy (FM). (A and D) The extent of observed endosomal localization is indicated (Loc.). Vps4 interactions affected by the different mutations are listed. Int., numbers are based on the interactions in Figure 1A. (B) Endosomal recruitment of MIT-deleted Vps4 in the presence or absence of full-length Vps4 protein determined by subcellular fractionation and Western blot analysis (S, soluble; P, pellet). (C) Immunoprecipitation of Vps20-HA from detergent-solubilized membrane fractions. The resulting bound and unbound samples were analyzed by Western blot using anti-Vps20 antiserum. (D) Fluorescence microscopy (FM) analysis of GFP-tagged full-length Vps4 protein in different mutant strains (Table 1). Numbers in parentheses indicate partially disrupted interactions.
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Related In: Results  -  Collection

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Figure 4: Localization of mutant Vps4 proteins. (A) Localization of MIT-deleted Vps4 (GFP- Vps4ΔMIT,E233Q and GFP-Vps4ΔMIT,E233Q,S377A) in different yeast mutant strains (see Table 1) determined by fluorescence microscopy (FM). (A and D) The extent of observed endosomal localization is indicated (Loc.). Vps4 interactions affected by the different mutations are listed. Int., numbers are based on the interactions in Figure 1A. (B) Endosomal recruitment of MIT-deleted Vps4 in the presence or absence of full-length Vps4 protein determined by subcellular fractionation and Western blot analysis (S, soluble; P, pellet). (C) Immunoprecipitation of Vps20-HA from detergent-solubilized membrane fractions. The resulting bound and unbound samples were analyzed by Western blot using anti-Vps20 antiserum. (D) Fluorescence microscopy (FM) analysis of GFP-tagged full-length Vps4 protein in different mutant strains (Table 1). Numbers in parentheses indicate partially disrupted interactions.
Mentions: The Vps4 L64D mutation caused a severe GFP-CPS sorting defect indicating that the loss of all MIM1 interactions blocked Vps4 function (Figure 3A, 4). In contrast, the MIM2-interaction mutant Vps4I18D only partially inhibited the activity of Vps4 (Figure 3A, 3), which is consistent with the observed partial loss of MITI18D-GFP recruitment to endosomes (Figure 2, A and B, 10). However, single and double mutants of the ESCRT-III MIM2 motifs resulted in normal GFP-CPS trafficking, further supporting the notion that mutants with weak MIT-localization defects show correspondingly weak (or no) MVB-trafficking phenotypes (Figure 3A, 14–16).

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.

Show MeSH
Related in: MedlinePlus