1. Volume 14, Issue 1, Pages 50-61 (January 2008)
ESCRT-III Family Members Stimulate Vps4 ATPase Activity Directly or via Vta1 
Ishara F. Azmi, Brian A. Davies, Junyu Xiao, Markus Babst, Zhaohui Xu, David J. Katzmann 
Developmental Cell 
Volume 14, Issue 1, Pages (January 2008)
DOI: /j.devcel
Copyright © 2008 Elsevier Inc. Terms and Conditions

2. Figure 1
Vta1 Interactions with Core ESCRT-III and Accessory ESCRT-III Proteins
(A) In vitro binding analysis of Vta1 with core ESCRT-III and accessory ESCRT-III subunits. Purified Vta1 was incubated with affinity-purified GST or GST-tagged Vps60, Did2, Snf7, Vps2, Vps20, and Vps24. Bound materials were visualized by western blotting with anti-Vta1 and Coomassie blue staining.
(B) Determinants impacting Vta1 localization to endosomal compartments. Fluorescence microscopy was utilized on wild-type and indicated deletion strains coexpressing Vta1-GFP and DsRed-FYVE.
Developmental Cell  , 50-61DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

3. Figure 2 Mapping Vta1 Binding Regions in Vps60 and Did2
(A) Schematic of predicted Vps60 α helices and truncations tested for binding to Vta1. Residues 144–148 are highlighted with an asterisk.
(B) In vitro binding analysis of Vps60 truncations and Vta1. GST pull-down assay was performed with GST, full-length Vps60, and corresponding Vps60 truncations and the point mutant. GST fusions were incubated with recombinant Vta1, and bound materials were visualized by western blotting with anti-Vta1 and Coomassie blue staining.
(C) Schematic of predicted Did2 α helices and truncations tested for binding to Vta1.
(D) In vitro binding analysis of Did2 truncations with Vta1. Bound materials were visualized by western blotting with anti-Vta1 and Coomassie blue staining.
Developmental Cell  , 50-61DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

4. Figure 3 Vps60 and Did2 Augment Vps4 ATPase Activity through Vta1
(A) Analysis of ESCRT-III subunits binding Vps4E233Q in the presence of ATP. GST or the indicated GST fusion proteins were incubated with purified Vps4E233Q, and bound material was visualized by Coomassie staining and western blotting with anti-Vps4 antibody.
(B) ATPase activity of Vps4 (0.5 μM) was measured with GST, GST-Vps60, GST-Did2, GST-Snf7, GST-Vps2, GST-Vps20, and GST-Vps24 in the presence or absence of Vta1 (2 μM). Activity is expressed as ADP/Vps4/min.
(C) ATPase activities of the wild-type and Vps4 lacking a MIT domain (Vps4ΔN) were measured with GST, GST-Vps60, GST-Did2, and GST-Vps2 in the presence or absence of Vta1.
Developmental Cell  , 50-61DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

5. Figure 4
Domain Analyses of Vps60 and Did2 Regions Critical for Stimulation of Vps4 ATPase Activity
(A) ATPase activities were measured with either GST or GST fusions corresponding to the α4-5 (128–184), α4 (128–159), and α5 (161–184) helices of Vps60 in the presence or absence of Vta1.
(B) ATPase activities of Vps4 were analyzed with GST, GST-Vps60, or GST-Vps60(144–148)Ala mutant in the presence or absence of Vta1.
(C) ATPase activities of wild-type Vps4 and Vps4ΔN were measured with GST, GST-Did2, and GST-Did2176–204 (C-terminal Did2 peptide) in the presence or absence of Vta1.
Developmental Cell  , 50-61DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

6. Figure 5
Vta1 MIT Regions Contribute to Vta1-Dependent Accessory ESCRT-III Stimulation of Vps4 ATPase Activity
(A) ATPase activities of wild-type Vps4 were measured with GST, GST-Vps60, and GST-Did2 in the presence of wild-type Vta1, Vta1 MIT2 mutants (Vta1W122A and Vta1K152A), or Vta1 lacking both MIT1 and MIT2 (Vta1165–330).
(B) ATPase activities of wild-type Vps4 were measured with GST, GST-Vps60 in the presence of wild-type Vta1, or a Vta1 MIT1 mutant (Vta1E57A).
Developmental Cell  , 50-61DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

7. Figure 6 In Vivo Analysis of Vta1 and Vps60 Mutants
(A) Cherry-Vps60 and Cherry-Vps60(144–148)Ala subcellular localizations were analyzed in the indicated genetic backgrounds.
(B) Cherry-Vps60 and Cherry-Vps60(144–148)Ala subcellular localizations were analyzed in vps60Δ vta1Δ cells expressing Vta1-GFP.
(C) CPS pulse-chase immunoprecipitation was performed to analyze the effects of Vta1 N-terminal mutations on CPS maturation. Percent CPS maturation was determined by comparing the amount of mature CPS to total CPS for each time point.
(D) Percent CPS maturation at 60 min was normalized to determine percent Vta1 function relative to wild-type.
(E) Subcellular fractionation was performed on lysates generated from vta1Δ cells or vta1Δ cells expressing wild-type Vta1, Vta1W122A, or Vta1K152A. Endogenous Snf7 was visualized by α-Snf7 antibody. α-PGK antibody was utilized as a marker for soluble fractions.
Developmental Cell  , 50-61DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions

8. Figure 7 Model of ESCRT-III Recruitment and Dissociation by Vps4-Vta1
(A) Schematic representation of Vps2, Vps4, Vps60, Did2, and Vta1, highlighting regions critical for interactions that contribute to increased Vps4 ATPase activity. The MIM (MIT interacting motif) in Vps2 and Did2 has recently been characterized as responsible for interacting with the MIT domain of Vps4 and is included for clarity (Obita et al., 2007; Stuchell-Brereton et al., 2007).
(B) Stepwise recruitment of Vps20-Snf7 “1” and Vps2-Vps24 “2” provides a docking site for Vps4-Vta1. This association allows for stimulation of Vps4 ATPase activity by Vps2 and dissociation of the core ESCRT-III subunits A and may represent the major mode of Vps4 activation (based on the relatively minor phenotypes observed upon loss of Vps60 or Did2). Dissociation step A may not require Vta1 function, as Vps2 can stimulate Vps4 directly. Did2 recruitment by Vps2-Vps24 “3” represents a second point at which Vps4 ATPase activity can be stimulated through Vta1, leading to dissociation step B. Last, recruitment of Vps60 by Vta1 can lead to dissociation step C. At present, the precise function of dissociation steps B and C are not entirely clear, but they appear to be required for maximal function of the MVB pathway. Several possible scenarios can be invoked as explanations for these three modes of dissociation, including a specific response to environmental or growth conditions, a “proofreading” activity by Vps4 to prevent inappropriate assembly of ESCRT-III, or distinct sites of action within the cell.
Developmental Cell  , 50-61DOI: ( /j.devcel )
Copyright © 2008 Elsevier Inc. Terms and Conditions