Structural role of the Vps4-Vta1 interface in ESCRT-III recycling

Abstract

The ESCRT complexes are required for multivesicular body biogenesis, macroautophagy, cytokinesis, and the budding of HIV-1. The final step in the ESCRT cycle is the disassembly of the ESCRT-III lattice by the AAA+ ATPase Vps4. Vps4 assembles on its membrane-bound ESCRT-III substrate with its cofactor, Vta1. The crystal structure of the dimeric VSL domain of yeast Vta1 with the small ATPase and the betadomains of Vps4 was determined. Residues involved in structural interactions are conserved and are required for binding in vitro and for Cps1 sorting in vivo. Modeling of the Vta1 complex in complex with the lower hexameric ring of Vps4 indicates that the two-fold axis of the Vta1 VSL domain is parallel to within approximately 20 degrees of the six-fold axis of the hexamer. This suggests that Vta1 might not crosslink the two hexameric rings of Vps4, but rather stabilizes an array of Vps4-Vta1 complexes for ESCRT-III disassembly.

Fig. 1. Binding of Vps4 and Vta1 constructs and their mutants

A. Binding curves from best fits to equilibrium binding model. B-I. SPR sensorgrams for the indicated constructs.

Fig. 2. Structure of the Vps4-Vta1 molecular interface

A. Electron density calculated from a 2Fo-Fc synthesis in which the residues shown in orange were omitted. Density is contoured at 1 σ. B. Ribbon model for the 1:1 complex in the asymmetric unit. C. Interaction of Vta1 with the molecular surface of Vps4, with the latter colored by atom type (carbon, green; oxygen, red; nitrogen, blue).

Fig. 3. The Vps4-Vta1 interacting complex is a dimer

Vta1 is shown in orange and magenta, Vps4 in cyan and blue. The view is looking down the two-fold crystallographic axis related the two Vta1-Vps4 complexes.

Fig. 4. Structure based alignment of interface residues

Residues in Vta1 (A) and Vps4 (B) are marked by asterisks where they are involved in direct interactions.

Fig. 5. Details of the interface

A. Ribbon model of the interface, with Vta1 in orange and Vps4 in blue. B. Surface representation of Vps4 colored by atom type, with Vta1 shown in a ribbon (orange) and ball-and-stick representation. C. Surface representation of Vta1 colored by atom type, with Vps4 shown in a ribbon (blue) and ball-and-stick representation.

Fig. 6. Structural interface residues are required for function

The indicated plasmids were transformed into vta1Δ yeast cells and imaged for GFP-Cps1 (cargo) and FM4-64 (membrane) fluorescence as shown. The scale bar is 5 μm.

Fig. 7

A model for cross-linking of Vps4 hexamers by the Vta1-VSL domain. A. Following superposition of the Vps4 SAB domain on one subunit of the Vps4 hexamer (green), the two-fold axis of the Vta1-VLS:Vps4-SAB complex (magenta) is within 23° of the six-fold axis of the hexamer. Part of the second Vps4-SAB overlaps with another subunit in the hexamer. B. Model for a putative unkinked conformation of α8. This model rotates the Vps4 β-domain and Vta1 by 40°, eliminates the steric overlap between the second SAB fragment and the hexamer, and is compatible with continuous hexagonal lattice packing. C. Hypothetical p6 lattice arrangement of Vta1-VSL (red) cross-linked Vps4 lower ring hexamers. For illustration purposes, a model of the human VPS4B hexamer is used instead of yeast, as the longer β6- β7 loop in human makes the orientation of the β domain easier to visualize.