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, 12 (6), 973-86

The Vps27/Hse1 Complex Is a GAT Domain-Based Scaffold for Ubiquitin-Dependent Sorting


The Vps27/Hse1 Complex Is a GAT Domain-Based Scaffold for Ubiquitin-Dependent Sorting

Gali Prag et al. Dev Cell.


The yeast Vps27/Hse1 complex and the homologous mammalian Hrs/STAM complex deliver ubiquitinated transmembrane proteins to the ESCRT endosomal-sorting pathway. The Vps27/Hse1 complex directly binds to ubiquitinated transmembrane proteins and recruits both ubiquitin ligases and deubiquitinating enzymes. We have solved the crystal structure of the core responsible for the assembly of the Vps27/Hse1 complex at 3.0 A resolution. The structure consists of two intertwined GAT domains, each consisting of two helices from one subunit and one from the other. The two GAT domains are connected by an antiparallel coiled coil, forming a 90 A-long barbell-like structure. This structure places the domains of Vps27 and Hse1 that recruit ubiquitinated cargo and deubiquitinating enzymes close to each other. Coarse-grained Monte Carlo simulations of the Vps27/Hse1 complex on a membrane show how the complex binds cooperatively to lipids and ubiquitinated membrane proteins and acts as a scaffold for ubiquitination reactions.


Figure 1
Figure 1. Modular Organization of Vps27 and Hse1 and Related Proteins, and Alignment of GAT Domains
(A) Modular organization of Vps27, Hse1, and other GAT-domain containing proteins. Domain name abbreviations are as follows: VHS, Vps27/Hrs/STAM; UIM, ubiquitin-interacting motif; SH3, Src homology-3; GAT, GGA and TOM; GGA, Golgi-localized, gamma-ear containing, ADP-ribosylation-factor-binding protein; TOM, target of Myb; FYVE, Fab1/YOTP/Vac1/EEA1; CB, clathrin-binding; DUIM, double UIM; NGAT, the N-terminal region preceding GAT domain, responsible for binding to Arf1-GTP; GAE, γ-adaptin ear. A helical region of Hrs is a putative, but unproven, GAT domain. (B) GAT domains were aligned based on three-dimensional structural superposition where available (Vps27, Hse1, GGA1, GGA3, and Tom1), and otherwise by sequence homology with the most similar protein of known structure. Colored dots or triangles indicate residues of Vps27 (blue) and Hse1 (orange) that participate in the heterodimer interface. Residues shown in triangles were mutated in this study. The letter “A” above the alignment denotes residues of Vps27 mutated to Ala (Bilodeau et al., 2003). The major site 1 ubiquitin-binding motif of GGA1, GGA3, and Tom1 as discussed in the text is outlined in black.
Figure 2
Figure 2. Hydrodynamic Properties of the Vps27/Hse1 Core Complex
(A) Gel filtration analysis of the recombinant Vps27/Hse1 core showing co-migration at an apparent mass of 29 kDa. This is slightly higher than the calculated mass of 23.2 kDa for a 1:1 complex, and is therefore consistent with an elongated 1:1 complex, but is not consistent with any oligomer with a greater number of subunits. (B) Sedimentation equilibrium profiles at 4.0°C plotted as a distribution of the absorbance at 280 nm vs. r at equilibrium. Data were collected at 13 (orange), 16 (yellow), 19 (green), 22 (cyan), 25 (blue) and 28 (brown) krpm at a loading A280 of 0.75. The solid lines show the best-fit global analysis (carried out for the three loading concentrations) in terms of a single ideal solute, with the corresponding residuals shown in the panels above the plot. (C) Sedimentation equilibrium profile at 4.0°C and 22 krpm plotted in terms of lnA280 vs. r2. The data shown correspond to a loading A280 of 0.25. The solid line indicates the plot expected for a monodisperse 1:1 Vps27:Hse1 complex. (D) Sedimentation equilibrium profile of the Vps27 core region in isolation at 4.0°C and 22 krpm plotted in terms of lnA280 vs. r2. The data shown correspond to a loading A280 of 0.60. The solid line indicates the plot expected for a monomeric Vps27, indicating the presence of higher oligomers.
Figure 3
Figure 3. Crystal Structure of the Vps27/Hse1 Complex
(A) Density-modified MAD Fourier synthesis (green) contoured at 1.0 σ and Se anomalous difference Fourier (red) contoured at 4.0 σ superimposed on the refined structure, with SeMet residues highlighted. (B) Overall structure of the core heterodimer, with Vps27 in blue and Hse1 in orange. (C) Superposition of the core portions of the Vps27 and Hse1 monomers. (D) Surface of Hse1 showing interactions with labeled residues of Vps27. (E) Surface of Vps27 showing interactions with labeled residues of Hse1.
Figure 4
Figure 4. GAT Domains in Vps27 and Hse1
(A) The HHV helical bundle. (B) The VVH helical bundle. (C) The GAT domain of Tom1 shown in the same orientation as in parts A and B. (D) Superposition of the HHV and VVH bundles and the Tom1 GAT domain. (E) Model for the closed monomeric conformation of the Vps27 GAT domain, generated by superimposing the Vps27 structure on the Tom1 GAT domain monomer.
Figure 5
Figure 5. The Vps27/Hse1 Interface is Required for Sorting
(A) Expression levels and co-immunoprecipitation of HA-tagged Vps27 and myc-tagged Hse1 proteins. Left panels. Whole cell lysates from hse1Δ vps27Δ cells co-transformed with Hse1-myc and Vps27-HA constructs were lysed and subjected to SDS-PAGE and immunoblot analysis with anti-HA and anti-myc antibodies. Right panels. Rabbit Anti-HA immunoprecipitation from the above lysates, followed by SDS-PAGE and immunoblotting with mouse anti-HA and mouse anti-myc antibodies. (B-I) Fluorescence microscopy images of GFP chimeras of Ste3 and Cps1 in vps27Δ cells expressing Vps27-WT or the indicated core complex mutants. (J) CPY maturation in hse1Δ vps27Δ cells expressing various constructs. Cells were metabolically labeled with 35S-metionine for 10 min (pulse), chased for 15 min in complete medium, and endogenous CPY was immunoprecipitated with anti-CPY antibody. The 15 min point samples were analyzed by SDS-PAGE followed by fluorography. (K) CPY colony blot assay on hse1Δ vps27Δ cells co-expressing Hse1-WT and various Vps27 constructs. Colonies from each strain were spotted onto selective medium and overlayed with nitrocellulose. Secreted CPY was detected by immunoblotting the nitrocellulose with an anti-CPY antibody.
Figure 6
Figure 6. A Unified Model for the Interaction of Vps27/Hse1 with Membranes and Ubiquitinated Cargo
The figure shows a single snapshot from the MC simulation. (A) View looking directly down towards the membrane. (B) View normal to the plane of the membrane (membrane surface indicated by the green line). Vps27 is shown in blue, Hse1 in orange, and ubiquitinated cargo in red.
Figure 7
Figure 7. Dynamics of the Vps27/Hse1 complex
(A) Distributions of the distances between the SH3 domain and three Ub-Cps1molecules. (B) Time series of the distance between the SH3 domain and the Hse1 UIM. (C) Distributions of the distances between the SH3 domain and three UIMs. (D) Average of Rg as a function of the overall system size. (E) Distributions of Rg for different system sizes. (F) Distributions of the distances between three UIMs and the membrane surface. (G) Fraction of Ub-Cps1 bound to the different UIMs (in isolation, on Vps27 alone, and on the full Hse1/Vps27 complex) as a function of the two-dimensional Ub-Cps1 concentration in the membrane.

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