The roles of Hrd1p ubiquitin ligase complex members Usa1p and Hrd3p in ER-associated degradation and Hrd1p regulation

Elimination of misfolded proteins from the endoplasmic reticulum (ER) is carried out by the highly conserved quality control pathway ER-associated degradation (ERAD). Misfolded proteins are targeted for destruction at the proteasome by covalent modification with a polyubiquitin chain. Hrd1p is a key ERAD ubiquitin ligases involved in mediating ubiquitination of misfolded proteins in S. cerevisiae. The action of Hrd1p requires several additional cofactors, including Hrd3p, a partner protein required for the stability of Hrd1p, and together constituting the core of the HRD complex. I investigated the ERAD functions of both Hrd3p and the additional complex member Usa1p, in order to better understand the mechanisms of HRD complex action in ERAD. These studies have found that Usa1p is a bipartite regulator of Hrd1p, promoting its degradation in the absence of Hrd3p and also affecting Hrd1p activity. The study of Usa1p has broadened our understanding of Hrd1p self-ubiquitination, which I demonstrated to proceed through a trans-ubiquitination mechanism. Improved understanding of Hrd1p degradation has resulted in a yeast strain in which Hrd1p stability is genetically uncoupled from substrate degradation in the absence of Hrd3p. This allowed explicit confirmation of the direct role of Hrd3p in ERAD. I have also uncovered an antagonistic relationship between Usa1p and Hrd3p, which has been suggested by their dichotomous roles in promoting Hrd1p stability and degradation. Over-expression of Hrd1p causes an activity dependent growth deficit in yeast, which is alleviated by over-expression of Usa1p. Addition of Hrd3p to this system restored the growth deficit, implying that Hrd3p and Usa1p have opposing actions in participating in or controlling Hrd1p ligase activity. These studies have provided significant insight into the mechanisms of the HRD complex, and have unveiled useful tools in which to further understand the Hrd3p's direct ERAD functions and the relationship between Hrd1p, Hrd3p, and Usa1