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. 2012 Mar 9;287(11):8633-40.
doi: 10.1074/jbc.M111.317644. Epub 2012 Jan 18.

Structural and Biochemical Basis of Yos9 Protein Dimerization and Possible Contribution to Self-Association of 3-hydroxy-3-methylglutaryl-coenzyme A Reductase Degradation Ubiquitin-Ligase Complex

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Structural and Biochemical Basis of Yos9 Protein Dimerization and Possible Contribution to Self-Association of 3-hydroxy-3-methylglutaryl-coenzyme A Reductase Degradation Ubiquitin-Ligase Complex

Jennifer Hanna et al. J Biol Chem. .
Free PMC article

Abstract

In yeast, the membrane-bound HMG-CoA reductase degradation (HRD) ubiquitin-ligase complex is a key player of the ER-associated protein degradation pathway that targets misfolded proteins for proteolysis. Yos9, a component of the luminal submodule of the ligase, scans proteins for specific oligosaccharide modifications, which constitute a critical determinant of the degradation signal. Here, we report the crystal structure of the Yos9 domain that was previously suggested to confer binding to Hrd3, another component of the HRD complex. We observe an αβ-roll domain architecture and a dimeric assembly which are confirmed by analytical ultracentrifugation of both the crystallized domain and full-length Yos9. Our binding studies indicate that, instead of this domain, the N-terminal part of Yos9 including the mannose 6-phosphate receptor homology domain mediates the association with Hrd3 in vitro. Our results support the model of a dimeric state of the HRD complex and provide first-time evidence of self-association on its luminal side.

Figures

FIGURE 1.
FIGURE 1.
Constructs of Yos9 used in this study. Domain organization of the full-length Yos9 protein (top) and its truncated variants (bottom) are shown. Numbers indicate the position of the respective amino acid. S, signal sequence for ER import; N, N-terminal region; C, C-terminal domain. The signal sequence and the last three residues were omitted from the protein hereinafter considered as full-length Yos9 (Yos924–539). Constructs that are solubly expressed in E. coli are marked with an asterisk.
FIGURE 2.
FIGURE 2.
Structure of the dimerization domain of Yos9. A, schematic representation of structure (Yos9266–424) from two different viewpoints related by 90° rotation around the vertical axis. Coloring follows the primary structure from blue (N terminus) to red (C terminus). α-Helices, β-strands, and extended loops are depicted as α1/2, β1–7, and L1–4, respectively. B, topology of Yos9 DD. Coloring corresponds to A.
FIGURE 3.
FIGURE 3.
Possible assemblies of Yos9266–424 in crystal structure. Coloring of the interfacing residues corresponds to Fig. 2, noninterfacing residues are grayed out. Upper, assemblies; lower, close-up view of interfaces. A, dimer of Yos9266–424 as present in the asymmetric unit. The extended N terminus of the upper molecule was omitted for clarity. The Greek key loop L4 covers the bottom of the β-sandwich. Side chains of mutated residues (Asn380, Leu393) are depicted in stick representation along with side chains of contacting residues. B, trimeric assembly at the crystallographic 3-fold axis. Side chains involved in hydrogen-bonding interactions are shown.
FIGURE 4.
FIGURE 4.
Yos9 self-association experiments. A, cross-linking of wild-type Yos9266–424. The protein was cross-linked at concentrations ranging from 1 to 20 μm with a 50-fold molar excess of bis[sulfosuccinimidyl] suberate (BS3). An untreated protein sample was run in the leftmost lane. B, sedimentation equilibrium results. The mean apparent molecular mass (Mapp) divided by the monomer molecular mass (Mmono) is plotted against the concentration. Crosses, Yos924–539; triangles, wild-type Yos9266–424; circles, Yos9266–424 L393A; squares, Yos9266–424 N380A.
FIGURE 5.
FIGURE 5.
Co-immunoprecipitation of Yos9 truncation variants. Microsomal extracts from a Yos9 knock-out strain expressing HA-tagged Hrd3 were immunoprecipitated in the presence of oligohistidine-tagged Yos9 truncation constructs. Co-immunoprecipitated Yos9 was detected by immunoblotting with anti-His antibody. Amino acid boundaries and domains included in the constructs are depicted on top of the corresponding lanes. Expected masses for the His7 fusion proteins are: Yos924–539, 62 kDa; Yos990–262, 23 kDa; Yos924–262, 30 kDa; Yos924–424, 49 kDa; Yos990–424, 41 kDa; Yos9266–539, 34 kDa; Yos9266–424, 21 kDa; and Yos924–90, 11 kDa.
FIGURE 6.
FIGURE 6.
Schematic model of self-association sites within the HRD complex. Brown arrows indicate association between two proteins, black arrows indicate self-association of the respective component. Self-associating proteins and known self-associating domains therein are highlighted by coloring. H/U, segments within the N-terminal cytosolic domain of Usa1 that are associated with Hrd1 interaction (H) and self-association (U), respectively (23).

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