Investigation of the regulation of exocytosis and endocytosis pathways in "Saccharomyces cerevisiae"

Polarized growth and remodeling of the plasma membrane proteome in response to environmental changes in yeast depends on regulated exocytosis and endocytosis. The yeast chitin synthase III, Chs3, shuttles between internal compartments and the plasma membrane to allow its cell cycle-dependent expression at the bud neck and uniform discharge at the cell surface upon heat stress. The exomer complex, comprised of Chs5 and the ChAP family of cargo recognition subunits, mediates the direct, controlled export of Chs3 from the trans-Golgi network (TGN) to the plasma membrane. To further establish the role of exomer in regulated trafficking, we characterized a novel exomer-dependent cargo, the prion-domain containing protein, Pin2.
The Pin2 cytosolic domain encompasses an exomer-binding site, located within the C-terminal prion domain, and most likely another interaction site towards the N-terminal region. In parallel, we found that a vast portion of the ChAP Chs6, required for Chs3 export, confers Chs3 specificity, suggesting a proportionally large binding surface on the cargo.
Pin2, like Chs3, localizes to the plasma membrane in a polarized, cell cycle-dependent manner. Moreover Pin2 and Chs3 share several trafficking requirements. Apart from exomer-mediated export, Pin2 and Chs3 undergo active recycling through endocytosis and clathrin adaptor complex 1 (AP-1)-mediated retrograde transport from early endosomes to the TGN. Recognition of AP-1 and most likely of the AP-2 endocytic adaptor could occur through a tyrosine rich YGENYYY sequence in Pin2. The active shuttling of Pin2 between the TGN, early endosomes and the plasma membrane is required for the polarized localization of Pin2 and seems to allow its immediate, stress-responsive redistribution. Upon lithium treatment Pin2 is rapidly endocytosed and maintained in internal compartments. Stress relief results in fast re-export of Pin2 to the plasma membrane.
The Pin2 prion domain contains the exomer and potential AP-1/AP-2 binding motifs. Therefore aggregation of this region may modify the interaction of Pin2 with sorting machineries. Indeed, we found that polarized localization and maintenance of Pin2 in internal compartments is compromised in a Pin2(QNtoED), prion domain mutant. Mutation of QN residues to charged amino acids in Pin2(QNtoED) inhibits the formation of SDS-resistant prion aggregates upon overexpression.
Reversible posttranslational modifications contribute an additional level of Pin2 trafficking regulation. Ubiquitylation of Pin2 is required for its endocytosis under physiological conditions and seems to play a crucial role in Pin2 internalization upon lithium stress. Modification within a cluster of four cytosolic cysteines by palmitoylation seems to support Pin2 cell surface expression. Interestingly, the presence of two luminal cysteines, which engage in the formation of disulfide-linked pin structure, is crucial for Pin2 export. Together this data demonstrates that several cytosolic motifs and the Pin2 prion domain, as well as a defined luminal structure, determine the regulated trafficking of Pin2.