Abstract

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a protein quality-control pathway in eukaryotes in which misfolded ER proteins are polyubiquitylated, extracted and ultimately degraded by the proteasome. This process involves ER membrane-embedded ubiquitin E2 and E3 enzymes, as well as a soluble E2 enzyme (Ubc7 in Saccharomyces cerevisiae and UBE2G2 in mammals). E2-binding regions (E2BRs) that recruit these soluble ERAD E2s to the ER have been identified in humans and S. cerevisiae, and structures of E2-E2BR complexes from both species have been determined. In addition to sequence and structural differences between the human and S. cerevisiae E2BRs, the binding of E2BRs also elicits different biochemical outcomes with respect to E2 charging by E1 and E2 discharge. Here, the Schizosaccharomyces pombe E2BR was identified and purified with Ubc7 to resolve a 1.7 Å resolution co-crystal structure of the E2BR in complex with Ubc7. The S. pombe E2BR binds to the back side of the E2 as an α-helix and, while differences exist, it exhibits greater similarity to the human E2BR. Structure-based sequence alignments reveal differences and conserved elements among these species. Structural comparisons and biochemistry reveal that the S. pombe E2BR presents a steric impediment to E1 binding and inhibits E1-mediated charging, respectively.

Highlights

  • Eukaryotic membrane and secreted proteins are translated on and folded in the endoplasmic reticulum (ER; Smith et al, 2011)

  • While the H. sapiens and S. cerevisiae E2-binding regions (E2BRs) domains used for crystallization were mostly ordered, with only a few residues at the N-terminus of G2BR that did not have sufficient density to be modeled, the longer S. pombe Ubc7-binding region (U7BR) construct used for crystallization had 22 N-terminal residues that were not observed in electron density (Li et al, 2009; Das et al, 2009; Metzger et al, 2013)

  • Recruitment of a soluble E2 (Ubc7 in yeast and UBE2G2 in humans) to the endoplasmic reticulum is essential for many ubiquitylation events in ER-associated degradation (ERAD) (Zattas & Hochstrasser, 2015)

Read more

Summary

Introduction

Eukaryotic membrane and secreted proteins are translated on and folded in the endoplasmic reticulum (ER; Smith et al, 2011). ERAD requires specific E2 and E3 enzymes, including a conserved soluble E2: UBE2G2 (humans) or Ubc (Saccharomyces cerevisiae) (Smith et al, 2011; Ye & Rape, 2009; Hiller et al, 1996; Biederer et al, 1996; Tiwari & Weissman, 2001). These E2s are recruited to the ER by membrane-embedded proteins. Similar to human G2BR, S. pombe U7BR may inhibit E1-mediated E2 charging

Cloning
Protein expression and purification
Preparation of the Ubc7–U7BR complex
Crystallization and data collection
Structure determination and refinement
Transthiolation assay
Results and discussion
Ubc7–U7BR intermolecular contacts
Discussion
Funding information
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call