A post-ER degradation pathway that relies on protease-dependent internalization from the vacuolar membrane

Newly synthesized proteins of the secretory pathway are quality-controlled inside the endoplasmic reticulum (ER) and, if not properly folded, are retained. An exception are glycosylphosphatidylinositol-anchored proteins (GPI-APs) which can leave the ER even when misfolded and are routed to the vacuole/lysosome for degradation by largely unknown mechanisms linked to post-ER quality control. Using yeast as model organism, we show that Gas1*, an ER-exported misfolded GPI-AP, is diverted from the secretory pathway to endosomes for transport to the vacuole. However, Gas1* is not sorted into endosomal intraluminal vesicles but internalizes directly from the vacuolar membrane. There, the vacuolar protease Pep4, but not any other known vacuolar protease, is required for Gas1* internalization. Our data reveal novel and unexpected mechanisms for invaginations from the vacuolar membrane.\n\nHighlightsO_LIER-exited misfolded GPI-anchored proteins are routed to the vacuole via endosomes but do not internalize into intraluminal vesicles\nC_LIO_LIInternalization occurs directly from the vacuolar membrane into intravacuolar mobile structures\nC_LIO_LIInternalization from the vacuolar membrane depends on the proteolytic activity of the vacuolar protease Pep4\nC_LI