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,Anja Schütz,Franziska Zimmermann,Joachim Behlke,Thomas Sommer,Udo Heinemann

doi:10.1074/jbc.m111.317644

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.

Highlights

Self-association of the HMG-CoA reductase degradation (HRD) complex is important for its function in endoplasmic reticulum (ER) quality control, but the oligomeric state of the complex is still unclear
The human ortholog of Yos9, OS-9, relies on an intact mannose 6-phosphate receptor homology (MRH) domain to bind to the Hrd3 ortholog SEL1L [40]. In agreement with this observation, our experiments suggest that the N-terminal part of Yos9 including the MRH domain is necessary for binding to Hrd3
The binding might be mediated by amino acids 24 –90 in a manner where the interaction can only be detected when the MRH domain stabilizes the N-terminal domain

Summary

Background

Self-association of the HRD complex is important for its function in ER quality control, but the oligomeric state of the complex is still unclear. We report the crystal structure of the Yos domain that was previously suggested to confer binding to Hrd, another component of the HRD complex. Our binding studies indicate that, instead of this domain, the N-terminal part of Yos including the mannose 6-phosphate receptor homology domain mediates the association with Hrd 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. Hrd interacts with ER-associated protein degradation substrates regardless of their glycosylation state, whereas Yos scans bound substrates for their glycans We propose that this additional dimerization interface on the luminal side of the HRD complex contributes to the overall self-association of the complex (supplemental Tables S2 and S3)

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION