SEL1L Protein Critically Determines the Stability of the HRD1-SEL1L Endoplasmic Reticulum-associated Degradation (ERAD) Complex to Optimize the Degradation Kinetics of ERAD Substrates

Yasutaka Iida,Tsutomu Fujimori,Katsuya Okawa,Kazuhiro Nagata,Ikuo Wada,Nobuko Hosokawa

doi:10.1074/jbc.m110.215871

Abstract

The mammalian HRD1-SEL1L complex provides a scaffold for endoplasmic reticulum (ER)-associated degradation (ERAD), thereby connecting luminal substrates for ubiquitination at the cytoplasmic surface after their retrotranslocation through the endoplasmic reticulum membrane. In this study the stability of the mammalian HRD1-SEL1L complex was assessed by performing siRNA-mediated knockdown of each of its components. Although endogenous SEL1L is a long-lived protein, the half-life of SEL1L was greatly reduced when HRD1 is silenced. Conversely, transiently expressed SEL1L was rapidly degraded but was stabilized when HRD1 was coexpressed. This was in contrast to the yeast Hrd1p-Hrd3p, where Hrd1p is destabilized by the depletion of Hrd3p, the SEL1L homologue. Endogenous HRD1-SEL1L formed a large ERAD complex (Complex I) associating with numerous ERAD components including ERAD lectin OS-9, membrane-spanning Derlin-1/2, VIMP, and Herp, whereas transiently expressed HRD1-SEL1L formed a smaller complex (Complex II) that was associated with OS-9 but not with Derlin-1/2, VIMP, or Herp. Despite its lack of stable association with the latter components, Complex II supported the retrotranslocation and degradation of model ERAD substrates α1-antitrypsin null Hong-Kong (NHK) and its variant NHK-QQQ lacking the N-glycosylation sites. NHK-QQQ was rapidly degraded when SEL1L was transiently expressed, whereas the simultaneous transfection of HRD1 diminished that effect. SEL1L unassociated with HRD1 was degraded by the ubiquitin-proteasome pathway, which suggests the involvement of a ubiquitin-ligase other than HRD1 in the rapid degradation of both SEL1L and NHK-QQQ. These results indicate that the regulation of the stability and assembly of the HRD1-SEL1L complex is critical to optimize the degradation kinetics of ERAD substrates.

Highlights

Hrd1p is an unstable protein with six N-terminal transmembrane segments that is stabilized by formation of a stoichiometric complex with Hrd3p, a type-I transmembrane protein with a large luminal domain containing SEL1 repeats [8, 9]
The results indicated that elimination of the SEL1L peptide by RNA interference (RNAi)-mediated HRD1 silencing occurred post-translationally
Silencing SEL1L or HRD1 Alters the Sedimentation Rate of the HRD1-SEL1L-containing ER-associated degradation (ERAD) Complex—SEL1L and HRD1 are known to form a stoichiometric complex, and a large ERAD complex was generated by the association of various ERAD components including Derlin-1/2/3, Herp, VIMP, p97/valosine-containing protein (VCP), OS-9, and so on

Summary

Introduction

Hrd1p is an unstable protein with six N-terminal transmembrane segments that is stabilized by formation of a stoichiometric complex with Hrd3p, a type-I transmembrane protein with a large luminal domain containing SEL1 repeats [8, 9]. While Complex II does not cosediment with the complex containing Derlin-1/2, Herp, and VIMP, Complex II does support the retrotranslocation and degradation of model ERAD substrates of misfolded glycoprotein NHK and non-glycosylated NHK-QQQ [16, 40, 41]. Silencing SEL1L or HRD1 Alters the Sedimentation Rate of the HRD1-SEL1L-containing ERAD Complex—SEL1L and HRD1 are known to form a stoichiometric complex, and a large ERAD complex was generated by the association of various ERAD components including Derlin-1/2/3, Herp, VIMP, p97/VCP, OS-9, and so on (see Fig. 7, Complex I).

Results

Conclusion