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. 2008 Jun;14(6):902-13.
doi: 10.1016/j.devcel.2008.04.004.

Integrated structural model and membrane targeting mechanism of the human ESCRT-II complex

Young Jun Im  1 , James H Hurley
Affiliations

Integrated structural model and membrane targeting mechanism of the human ESCRT-II complex

Young Jun Im et al. Dev Cell. 2008 Jun.

Abstract

ESCRT-II plays a pivotal role in receptor downregulation and multivesicular body biogenesis and is conserved from yeast to humans. The crystal structures of two human ESCRT-II complex structures have been determined at 2.6 and 2.9 A resolution, respectively. The complex has three lobes and contains one copy each of VPS22 and VPS36 and two copies of VPS25. The structure reveals a dynamic helical domain to which both the VPS22 and VPS36 subunits contribute that connects the GLUE domain to the rest of the ESCRT-II core. Hydrodynamic analysis shows that intact ESCRT-II has a compact, closed conformation. ESCRT-II binds to the ESCRT-I VPS28 C-terminal domain subunit through a helix immediately C-terminal to the VPS36-GLUE domain. ESCRT-II is targeted to endosomal membranes by the lipid-binding activities of both the Vps36 GLUE domain and the first helix of Vps22. These data provide a unifying structural and functional framework for the ESCRT-II complex.

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Figures

Figure 1
Figure 1. Structure of human ESCRT-II complex
(A) Schematic of ordered regions visualized in human (this study) and yeast ESCRT-II (Hierro et al., 2004; Teo et al., 2004) complexes, with a comparison to the domain structures of intact human and yeast ESCRT-II complexes. (B) Overall structure of the complex. The WH2 domain of one of the VPS25 subunits was poorly visible in electron density map and it was not included in the structure refinement. The missing VPS25-WH2 was modeled in the figure using the structure of other subunit. (C) A top view of the complex showing a relatively flat “profile” of the complex. (D) Electron density from solvent-flattened MIR map contoured at 1.0 σ in the vicinity of VPS22–VPS36 portion of the core. The final refined structure was shown in a tube model. (E) Crystal structures in forms I and II are shown colored by B-factor to show regions of high (red) and low (blue) mobility.
Figure 2
Figure 2. The helical domain of ESCRT-II
(A) N-terminal helical domain (HD) of VPS22 and VPS36. (B) Comparison of human and yeast ESCRT-II in the HD region. The newly observed N-terminal helical domain formed by human VPS22 and VPS36 is shown at left, as compared to the isolated Vps22 fragment of this domain seen in the yeast structure at right.
Figure 3
Figure 3. Solution structure and ESCRT-I interactions of ESCRT-II
(A) Analysis of recombinant ESCRT-II complex on a Superdex 200 (16/60) column monitored by absorption at 280 nm. Comparison of the intact ESCRT-I Stokes radius RH derived from size exclusion data. The calculated RH values from crystal structures of the ESCRT-II constructs correspond precisely to the value expected from fitting to gel filtration standards. The standards are shown in open circles (BioRad, Hercules, CA) consist of bovine thyroglobulin (670 kDa, RH = 8.5 nm), bovine γ-globulin (158 kDa, RH = 5.3 nm), chicken ovalbumin (44 kDa, RH = 2.7 nm), and horse myoglobin (17 kDa, RH = 2.1 nm). (B) Solution conformation of intact ESCRT-II derived by fitting structural coordinates to hydrodynamic data from the four constructs shown in (A) using Hydropro (Garcia de la Torre et al., 2000). (C) Constructs used in this study. (D) GST-pull down experiment showing a direct interaction between VPS28-CTD and the various ESCRT-II constructs. The ESCRT-II constructs used for the assay are shown in lanes 2 – 8 for reference. The absence of binding of full length ESCRT-II to GST bound beads is shown in Lanes 9 and 10 as a control. Bands corresponding to subunits of complexes that are positive for VPS28-CTD binding are highlighted with dots colored as in panel (B).
Figure 4
Figure 4. Liposome binding of ESCRT-II complex
Purified constructs of the ESCRT-II complex were mixed with liposomes. (A) The ESCRT-II constructs used for liposome binding assay are shown for reference. All constructs used for the binding assay lack the VPS25-WH2 for expedience. However the binding of this construct and full-length ESCRT-II are essentially identical (Supplementary Fig. 3). (B) Binding to PC:PE liposomes. Molecular weight markers are shown in lane 1. Unbound samples in supernatants were shown in lanes 2 – 5 for reference. (C) – (J) Liposome binding results with different lipid compositions. Variable amounts of PI and PIPs were mixed to the PE : PC mixture to examine the specificity. The mole fractions of PIPs were chosen to maintain a constant charge density on the membrane. (K) The relative amounts of proteins in the pellets were shown in bars. The construct double deletion construct (c), which shows negligible binding to all liposomes tested, is not shown in bar graphs.
Figure 5
Figure 5. Cargo sorting and localization of Vps22 and Vps36 mutants
(A–L) The uppermost panel of each column shows the sorting of the GFP-Cps1 construct (green) in various strains, as indicated at the top of each column. (M–S) The localization of the designated ESCRT-II constructs in the indicated strains, as monitored by Vps22-GFP. The middle panels show the limiting membrane of the vacuole as labeled by FM4-22 64 (red), and the lower panels show the DIC image. Results presented here are characteristic of observations of > 100 cells for each strain shown.
Figure 6
Figure 6. Combinatorial membrane targeting of ESCRT-II
(A) Overall schematic representation of full length ESCRT-II structure showing the binding site for VPS28-CTD and the VPS22-H0 examined in this study, and the previously described binding sites for PI(3)P (Teo et al., 2006), ubiquitin (Alam et al., 2006; Hirano et al., 2006), and VPS20 (Langelier et al., 2006). (B) Model for combinatorial targeting by specific and nonspecific interactions with membrane (represented by the solid horizontal bar) lipids.
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