MISFOLDED WSC1P UNDERGO POST-ER QUALITY CONTROL

Abstract

Quality control (QC) mechanisms monitor the folding and assembly of newly synthesized proteins. The most well characterized QC pathway occurs in the ER and is termed ER quality control (ERQC) which targets misfolded proteins to be degraded via ER-associated degradation (ERAD). Post-ER QC pathways, albeit poorly understood, function to capture proteins that exit the ER prematurely. In our study, we reported a yeast plasma membrane protein Wsc1p to be a substrate that demonstrates the fundamental role of the Golgi in protein QC. A panel of Wsc1p variants misfolded in the extracellular/luminal domain was generated. The variants are degraded in an ERAD-independent pathway. Instead, they traffic to the Golgi from where they are delivered to the vacuole for degradation. Two reasons can account for the ERQC evasion of Wsc1p. First, a strong export signal in the cytoplasmic domain renders its efficient ER exit whether it is folded or not and whether it contains an ERAD determinant. Second, the luminal domain of Wsc1p lacks functional ERAD signals and a chaperone binding site. The identification and characterization of Wsc1p as an endogenous and obligate substrate reinforces the importance of the Golgi QC as a primary surveillance mechanism in the secretory pathway and provides a physiological basis for its existence.

Golgi QC generally recognizes misfolded proteins in the Golgi apparatus and targets them to the vacuole/lysosome for degradation. For misfolded membrane proteins, there are two fates. They can be localized to either the limiting vacuolar/lysosomal membrane or the lumen. To understand how Golgi QC delivers its misfolded membrane proteins to the vacuole, we examined Wsc1p variants with a misfolded luminal domain that are bona fide substrate of Golgi QC. We found that the mutants are transported from the Golgi to the vacuolar lumen via the multi-vesicular body (MVB) pathway. MVB sorting requires ubiquitination at the lysine residue(s) in the cytoplasmic domain of misfolded Wsc1p and the endosomal sorting complex required for transport (ESCRT) machinery. Most importantly, mislocalization of the variants at the limiting vacuolar membrane results in a series of degradation fragments suggesting incomplete elimination. This provides a physiological basis for the vacuolar lumen targeting of misfolded membrane substrates in Golgi QC. It ensures efficient degradation of the entire molecules and prevents the accumulation of potentially toxic fragments.