Endocytic membrane protein sorting and trafficking is essential to maintain the functional organizations and identities of different organelles. We studied this theme from three aspects: the ligand-stimulated down-regulation of plasma membrane protein (Chapter 1); the endosome-to-Golgi retrograde transport (Chapter 2) and the maintenance of Golgi-resident membrane proteins (Chapter 3).

When extracellular copper is added to the copper-starved cells, high affinity copper transporter Ctr1p is rapidly endocytosed, delivered to the lumen of the vacuole, and slowly degraded by vacuolar proteases. Two lysine residues in the C-terminal cytoplasmic tail of Ctr1p, Lys340 and Lys345, were found to be critical for copper-dependent endocytosis and degradation. In response to copper addition, Ctr1p was found to be ubiquitylated and a mutation in the Rsp5 ubiquitin ligase largely abolished ubiquitylation, endocytosis and degradation. In a strain lacking the Rsp5p accessory factors Bul1p and Bul2p, endocytosis and degradation of Ctr1p-GFP was substantially diminished.

Sorting nexin Mvp1p was reported to be involved in the vacuolar membrane protein sorting. Mvp1p needs the binding with PtdIns(3)P through PX domain for its endosomal localization and broadly localizes to the endosomal system. Mvp1p forms self-oligomerization like other BAR domain containing proteins. Mvp1p is required for acidic hydrolase receptor Vps10p endosome-to-Golgi retrograde transport. Vps10p-GFP is retained in endosomes in mvp1Δ mutant. Mvp1p might function to efficiently sort Vps10p to the retromer containing retrieval vesicles for retrograde transport.

The mechanism of glycosyltransferase localization to the Golgi apparatus is a long-standing question in secretory cell biology. All Golgi glycosyltransferases are type II membrane proteins with small cytosolic domains that contribute to Golgi localization. Yeast Vps74p directly binds to the cytosolic domains of cis and medial Golgi mannosyltransferases and that loss of this interaction correlates with loss of Golgi localization of these enzymes. Our collaborators have solved the X-ray crystal structure of Vps74p and found that it forms a tetramer, which is also observed in solution. Deletion of a critical structural motif disrupts tetramer formation, and results in loss of Vps74p localization and function.