Pep4-dependent microautophagy is required for post-ER degradation of GPI-anchored proteins

Abstract

Clearance of misfolded proteins from the secretory pathway often occurs soon after their biosynthesis by endoplasmic reticulum (ER)-associated protein degradation (ERAD). However, certain types of misfolded proteins are not ERAD substrates and exit the ER. They are then scrutinized by ill-defined post-ER quality control (post-ERQC) mechanisms and are frequently routed to the vacuole/lysosome for degradation. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) constitute a class of proteins of the secretory pathway that mostly depends on post-ERQC. How misfolded GPI-APs are detected, transported to the vacuole/lysosome and taken up by this organelle was poorly defined. Originating from the intriguing observation that several misfolded GPI-APs accumulate in the yeast vacuolar membrane in the absence of the major vacuolar protease Pep4, we designed an unbiased genome-wide screen in yeast and followed the trafficking of the misfolded fluorescent GPI-AP Gas1* from the ER to the vacuolar lumen. Our results reveal that post-ERQC of GPI-APs is linked with a novel type of microautophagy.

Figure 1.

A yeast post-ER degradation pathway links to Pep4-dependent microautophagy. GPI-APs are synthesized at the ER. They are devoid of cytosolic tails and TMDs and known to associate with sphingolipids and sterols in specific membrane domains (rafts). After ER export, correctly folded GPI-APs (blue) are routed to the plasma membrane (PM) via the secretory pathway. Misfolded GPI-APs (red) are diverted from the secretory pathway at the Golgi and are routed to endosomes by a process involving Pep1/Vps10. Unlike membrane-bound cargos that contain cytosolic tails and become ubiquitinated for ESCRT-mediated internalization from endosomes, forming multi-vesicular bodies (MVBs), misfolded GPI-APs fail to internalize at this stage. Instead, after endosome-vacuole fusion, they internalize from the vacuolar membrane into intravacuolar vesicles (IVVs). This microautophagic process is Pep4-dependent and negatively regulated by VTCs; it might be aided by raft-like properties in the vacuolar membrane. Subsequent degradation of IVVs depends on the vacuolar lipase Atg15.