UCSF

This work focuses on how the endoplasmic reticulum-associated degradation (ERAD) quality control machinery is able to distinguish terminally misfolded glycoproteins from folding intermediates and target these potentially toxic forms across the ER membrane for degradation. For luminal, glycosylated proteins, this discrimination depends both on the protein's folding status and on its glycosylation state, but much is still unknown about the mechanism.

My research centers on Yos9p, a recently identified protein that is essential for luminal ERAD in Saccharomyces cerevisiae. I demonstrated that Yos9p's predicted glycan-binding domain is necessary for ERAD and together with data showing that Yos9p interacts with ERAD substrates, these results implicated Yos9p as a critical sensor of the glycan-based degradation signal on misfolded substrates. Thus two important questions arose: how does Yos9p participate with the rest of the ERAD machinery to target substrates for degradation, and what is the glycan destruction signal that Yos9p recognizes? By affinity purifying Yos9p, I found that Yos9p, its binding partner, Hrd3p, and the chaperone Kar2p form the luminal components of a multi-protein complex that organizes key ERAD factors including the transmembrane ubiquitin ligase Hrd1p and cytosolic AAA ATPase Cdc48p. My characterization of this complex complemented the observation that Yos9p and Hrd3p each form a complex specifically with misfolded substrates independently of substrate glycosylation. My studies further suggested that Yos9p and Hrd3p act as gatekeepers that ensure that only legitimate substrates are degraded. Together, these findings indicate that Yos9p forms a luminal surveillance complex with Hrd3p to recruit misfolded proteins to the core ERAD complex and that Yos9p assists in a distinct sugar-dependent step necessary for degradation of correct substrates.

To characterize Yos9p's glycan specificity, I developed a protocol to make functional Yos9p from E. coli by refolding the protein in vitro. Frontal affinity chromatography determined that Yos9p recognizes N-linked glycans containing a terminal alpha 1,6-linked mannose. My in vivo studies then showed that this glycan signal is necessary for degradation. I also provide evidence that another ERAD factor, Htm1p, generates this specific glycan for recognition by Yos9p, adding another step to potentially increase the specificity of the recognition process.