ABSTRACT
We isolated a temperature-sensitive mutant, hrd4-1, deficient in ER-associated degradation (ERAD). The HRD4 gene was identical to NPL4, a gene previously implicated in nuclear transport. Using a diverse set of substrates and direct ubiquitination assays, our analysis revealed that HRD4/NPL4 is required for a poorly characterized step in ERAD after ubiquitination of target proteins but before their recognition by the 26S proteasome. Our data indicate that this lack of proteasomal processing of ubiquitinated proteins constitutes the primary defect in hrd4/npl4 mutant cells and explains the diverse set of hrd4/npl4 phenotypes. We also found that each member of the Cdc48p-Ufd1p-Npl4p complex is individually required for ERAD.
Subject(s)
Cysteine Endopeptidases/metabolism , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/metabolism , Multienzyme Complexes/metabolism , Nuclear Pore Complex Proteins , Nuclear Proteins/metabolism , Protein Processing, Post-Translational , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Ubiquitin/metabolism , Active Transport, Cell Nucleus , Adenosine Triphosphatases , Cell Cycle Proteins/metabolism , Fatty Acids, Unsaturated/metabolism , Flow Cytometry , Mutation , Nuclear Proteins/genetics , Nucleocytoplasmic Transport Proteins , Proteasome Endopeptidase Complex , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Valosin Containing Protein , Vesicular Transport ProteinsABSTRACT
In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin-proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin-protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro - catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.
Subject(s)
Endoplasmic Reticulum/metabolism , Intracellular Membranes/metabolism , Ligases/metabolism , Proteins/metabolism , Saccharomyces cerevisiae Proteins , Ubiquitin-Conjugating Enzymes , Ubiquitins/metabolism , Catalysis , Cysteine Endopeptidases/metabolism , Endoplasmic Reticulum/ultrastructure , High Mobility Group Proteins/metabolism , Intracellular Membranes/ultrastructure , Multienzyme Complexes/metabolism , Phenotype , Proteasome Endopeptidase Complex , Protein Structure, Tertiary/physiology , Ubiquitin-Protein Ligases , YeastsABSTRACT
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.