Endoplasmic Reticulum Degradation Requires Lumen to Cytosol Signaling

Endoplasmic reticulum (ER)-associated degradation (ERAD) is required for ubiquitin-mediated destruction of numerous proteins. ERAD occurs by processes on both sides of the ER membrane, including lumenal substrate scanning and cytosolic destruction by the proteasome. The ER resident membrane proteins Hrd1p and Hrd3p play central roles in ERAD. We show that these two proteins directly interact through the Hrd1p transmembrane domain, allowing Hrd1p stability by Hrd3p-dependent control of the Hrd1p RING-H2 domain activity. Rigorous reevaluation of Hrd1p topology demonstrated that the Hrd1p RING-H2 domain is located and functions in the cytosol. An engineered, completely lumenal, truncated version of Hrd3p functioned normally in both ERAD and Hrd1p stabilization, indicating that the lumenal domain of Hrd3p regulates the cytosolic Hrd1p RING-H2 domain by signaling through the Hrd1p transmembrane domain. Additionally, we identified a lumenal region of Hrd3p dispensable for regulation of Hrd1p stability, but absolutely required for normal ERAD. Our studies show that Hrd1p and Hrd3p form a stoichiometric complex with ERAD determinants in both the lumen and the cytosol. The HRD complex engages in lumen to cytosol communication required for regulation of Hrd1p stability and the coordination of ERAD events on both sides of the ER membrane.

[1]  D. Y. Thomas,et al.  Saccharomyces cerevisiae CNE1 Encodes an Endoplasmic Reticulum (ER) Membrane Protein with Sequence Similarity to Calnexin and Calreticulin and Functions as a Constituent of the ER Quality Control Apparatus (*) , 1995, The Journal of Biological Chemistry.

[2]  S. Emr,et al.  A membrane‐associated complex containing the Vps15 protein kinase and the Vps34 PI 3‐kinase is essential for protein sorting to the yeast lysosome‐like vacuole. , 1993, The EMBO journal.

[3]  T. Hunter,et al.  The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. , 1999, Science.

[4]  T. Biederer,et al.  Degradation of subunits of the Sec61p complex, an integral component of the ER membrane, by the ubiquitin‐proteasome pathway. , 1996, The EMBO journal.

[5]  A. Varshavsky,et al.  The E2–E3 interaction in the N‐end rule pathway: the RING‐H2 finger of E3 is required for the synthesis of multiubiquitin chain , 1999, The EMBO journal.

[6]  P. Edwards,et al.  Mevalonolactone inhibits the rate of synthesis and enhances the rate of degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in rat hepatocytes. , 1983, The Journal of biological chemistry.

[7]  M. A. Alonso,et al.  Intracellular retention and degradation of human mutant variant of a alpha 1-antitrypsin in stably transfected Chinese hamster ovary cell lines. , 1993, European journal of biochemistry.

[8]  J. Lippincott-Schwartz,et al.  Degradation from the endoplasmic reticulum: Disposing of newly synthesized proteins , 1988, Cell.

[9]  M. Peter,et al.  Ubiquitin-dependent degradation of multiple F-box proteins by an autocatalytic mechanism. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Satoshi Omura,et al.  Degradation of CFTR by the ubiquitin-proteasome pathway , 1995, Cell.

[11]  R. Hampton,et al.  Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Peter Walter,et al.  Functional and Genomic Analyses Reveal an Essential Coordination between the Unfolded Protein Response and ER-Associated Degradation , 2000, Cell.

[13]  L. Chan,et al.  Ubiquitin-proteasome pathway mediates intracellular degradation of apolipoprotein B. , 1996, Biochemistry.

[14]  R. D. Simoni,et al.  The regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase requires a short-lived protein and occurs in the endoplasmic reticulum. , 1990, The Journal of biological chemistry.

[15]  R. Plemper,et al.  Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation. , 1999, Journal of cell science.

[16]  M. Knop,et al.  Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast. , 1993, European journal of biochemistry.

[17]  D. Wolf,et al.  ER Degradation of a Misfolded Luminal Protein by the Cytosolic Ubiquitin-Proteasome Pathway , 1996, Science.

[18]  R. Gardner,et al.  HRD gene dependence of endoplasmic reticulum-associated degradation. , 2000, Molecular biology of the cell.

[19]  Jörg Urban,et al.  A regulatory link between ER-associated protein degradation and the unfolded-protein response. , 2000, Nature Cell Biology.

[20]  Shengyun Fang,et al.  Mdm2 Is a RING Finger-dependent Ubiquitin Protein Ligase for Itself and p53* , 2000, The Journal of Biological Chemistry.

[21]  R. Schekman,et al.  The engagement of Sec61p in the ER dislocation process. , 1999, Molecular cell.

[22]  Y. Xiong,et al.  Association with cullin partners protects ROC proteins from proteasome-dependent degradation , 1999, Oncogene.

[23]  A. Bernstein,et al.  Cloning and characterization of Sel-1l, a murine homolog of the C. elegans sel-1 gene , 1998, Mechanisms of Development.

[24]  J. Rine,et al.  Sequence determinants for regulated degradation of yeast 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. , 1998, Molecular biology of the cell.

[25]  M. Nakanishi,et al.  Multivalent control of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Mevalonate-derived product inhibits translation of mRNA and accelerates degradation of enzyme. , 1988, The Journal of biological chemistry.

[26]  D. Wolf,et al.  A RING‐H2 finger motif is essential for the function of Der3/Hrd1 in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae , 1999, FEBS letters.

[27]  S. Ho,et al.  Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. , 1989, Gene.

[28]  S. Ōmura,et al.  The Degradation of Apolipoprotein B100 Is Mediated by the Ubiquitin-proteasome Pathway and Involves Heat Shock Protein 70* , 1997, The Journal of Biological Chemistry.

[29]  Y. Xiong,et al.  ROC1, a homolog of APC11, represents a family of cullin partners with an associated ubiquitin ligase activity. , 1999, Molecular cell.

[30]  G. R. Carson,et al.  Associations between subunit ectodomains promote T cell antigen receptor assembly and protect against degradation in the ER , 1993, The Journal of cell biology.

[31]  Y. Suzuki,et al.  Mutations in PEX10 is the cause of Zellweger peroxisome deficiency syndrome of complementation group B. , 1998, Human molecular genetics.

[32]  E. Fisher,et al.  Regulated Co-translational Ubiquitination of Apolipoprotein B100 , 1998, The Journal of Biological Chemistry.

[33]  S. Fang,et al.  RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[34]  J. Rine,et al.  Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein. , 1996, Molecular biology of the cell.

[35]  G. Bitter,et al.  Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3-phosphate dehydrogenase gene promoter. , 1984, Gene.

[36]  S. Emr,et al.  The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene , 1994, Cell.

[37]  K Nasmyth,et al.  Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34. , 1999, Genes & development.

[38]  J. Rine,et al.  In vivo examination of membrane protein localization and degradation with green fluorescent protein. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[39]  M. Knop,et al.  Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. , 1996, The EMBO journal.

[40]  A. Nakano,et al.  Identification of SEC12, SED4, truncated SEC16, and EKS1/HRD3 as multicopy suppressors of ts mutants of Sar1 GTPase. , 1999, Journal of biochemistry.

[41]  K. Kuchler,et al.  Endoplasmic Reticulum Degradation of a Mutated ATP-binding Cassette Transporter Pdr5 Proceeds in a Concerted Action of Sec61 and the Proteasome* , 1998, The Journal of Biological Chemistry.

[42]  J. Rine,et al.  Regulated degradation of HMG-CoA reductase, an integral membrane protein of the endoplasmic reticulum, in yeast , 1994, The Journal of cell biology.

[43]  F. Li,et al.  The hereditary renal cell carcinoma 3;8 translocation fuses FHIT to a patched-related gene, TRC8. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[44]  R. Plemper,et al.  Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. , 1998, Molecular biology of the cell.

[45]  I. Greenwald,et al.  Structure, function, and expression of SEL-1, a negative regulator of LIN-12 and GLP-1 in C. elegans. , 1997, Development.

[46]  J. Peters,et al.  Subunits and substrates of the anaphase-promoting complex. , 1999, Experimental cell research.

[47]  S. Elledge,et al.  Reconstitution of G1 cyclin ubiquitination with complexes containing SCFGrr1 and Rbx1. , 1999, Science.