![]() This interaction regulates the activity of the Hrd1p cytosolic RING-H2 domain to allow ERAD by preventing Hrd1p degradation. ![]() Importantly, we have clearly shown that the Hrd1p transmembrane domain alone mediates the interaction between Hrd1p and Hrd3p. From these studies, we have demonstrated for the first time a direct physical interaction between the two proteins, defined domain-specific functions for each protein, ascertained the correct topology of Hrd1p, demonstrated transmembrane communication between Hrd1p and Hrd3p, and evaluated the importance of the stoichiometry of the Hrd1p–Hrd3p complex. ![]() In this paper, we have performed an extensive study of the functional interplay between Hrd1p and Hrd3p. Although the Hrd1p NH 2-terminal transmembrane domain contains conserved sequences, no function has been suggested for this anchor. Unlike other family members, the Hrd1p RING-H2 domain is anchored to the ER membrane by an NH 2-terminal, multimembrane spanning domain. In fact, we have recently demonstrated that Hrd1p is a ubiquitin ligase, catalyzing the transfer of ubiquitin from Ubc7p to ERAD substrates by the activity of its RING-H2 motif, which is required for a direct in vivo physical interaction between Hrd1p and Ubc7p (our unpublished results). 1999), Apc11p ( Peters 1999), and c-Cbl ( Joazeiro et al. The Hrd1p RING-H2 motif is conserved in a group of ubiquitin ligases or E3 proteins, including Ubr1p ( Xie and Varshavsky 1999), Hrt1p/Rbx1p ( Ohta et al. Hrd1p is also an ER-associated protein with two distinct domains: an NH 2-terminal, hydrophobic region with multiple predicted transmembrane spans and a COOH-terminal, hydrophilic region containing a RING-H2 motif required for the function of Hrd1p in ER degradation ( Hampton et al. However, the interplay and specific actions of Hrd1p/Hrd3p are poorly defined in current models of ERAD. Our recent work demonstrates that Hrd1p provides the core ubiquitin ligase (E3) activity in ERAD (our unpublished results), recruiting Ubc7p for substrate ubiquitination. Unlike Ubc7p, the functions of Hrd1p and Hrd3p are not as clear. These studies also revealed two previously unknown membrane proteins, Hrd1p (also isolated as Der3p) and Hrd3p, required for the ER degradation of numerous proteins ( Hampton et al. 2000 and our unpublished results), sometimes in conjunction with other ubiquitin-conjugating enzymes ( Biederer et al. 1996 Hampton and Bhakta 1997 Wilhovsky et al. These studies indicate that the ER-associated ubiquitin-conjugating enzyme Ubc7p is centrally involved in the degradation of ER substrates ( Biederer et al. 1996 Hampton and Bhakta 1997 Bordallo et al. Genes involved in ERAD have been identified by several genetic analyses in yeast ( Hampton et al. 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. Our studies show that Hrd1p and Hrd3p form a stoichiometric complex with ERAD determinants in both the lumen and the cytosol. Additionally, we identified a lumenal region of Hrd3p dispensable for regulation of Hrd1p stability, but absolutely required for normal ERAD. 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. Rigorous reevaluation of Hrd1p topology demonstrated that the Hrd1p RING-H2 domain is located and functions in the cytosol. 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. The ER resident membrane proteins Hrd1p and Hrd3p play central roles in ERAD. ERAD occurs by processes on both sides of the ER membrane, including lumenal substrate scanning and cytosolic destruction by the proteasome. Endoplasmic reticulum (ER)-associated degradation (ERAD) is required for ubiquitin-mediated destruction of numerous proteins.
0 Comments
Leave a Reply. |