The genome of the yeast Saccharomyces cerevisiae encodes five R-SNAREs: Snc1/2p, Ykt6p, Sec22p and Nyv1p. Of these three, Sec22p, Ytk6p and Nyv1p have N-terminal extensions of their SNARE-motifs greater than 100 amino acids in length. The structures of the N-terminal domains of Sec22b (the orthologue of yeast Sec22p) and of Ytk6p have revealed that both proteins share a similar profilin-like fold. R-SNAREs that contain a profilin-like fold in their N-terminal domains have been termed longins. What are the functions of SNARE longin domains? My thesis research has focused on their possible roles in regulating the sorting of these SNAREs as well as in the role this domain plays in the steady-state distribution of these proteins in the cell....[ Read more ]

The genome of the yeast Saccharomyces cerevisiae encodes five R-SNAREs: Snc1/2p, Ykt6p, Sec22p and Nyv1p. Of these three, Sec22p, Ytk6p and Nyv1p have N-terminal extensions of their SNARE-motifs greater than 100 amino acids in length. The structures of the N-terminal domains of Sec22b (the orthologue of yeast Sec22p) and of Ytk6p have revealed that both proteins share a similar profilin-like fold. R-SNAREs that contain a profilin-like fold in their N-terminal domains have been termed longins. What are the functions of SNARE longin domains? My thesis research has focused on their possible roles in regulating the sorting of these SNAREs as well as in the role this domain plays in the steady-state distribution of these proteins in the cell.

In collaboration with Prof. Mingjie Zhang’s group, we established that the Nyv1p N-terminal domain also contains a longin fold. I find that the longin domain of Nyv1p is both necessary and sufficient for the sorting of this protein to the limiting membrane of the vacuole - a process that is mediated by the adaptin complex AP-3. Further, I identify an adaptin tyrosine-based sorting motif in the Nyv1p longin domain that I show is required for the interaction of Nyv1p with the AP-3 complex in vivo and in vitro. This research has been published (Wen, W., Chen, L., Wu, H., Sun, X., Zhang, M. and Banfield, D.K. (2006) Molecular Biology of the Cell 17: 4282 - 4299). Glutamine scanning mutagenesis of surface exposed residues in the login domain of Sec22p revealed the presence of an evolutionary conserved patch on the surface of its longin domain, which is necessary for re-cycling the protein from the Golgi to ER. I hypothesize that the folded-back cytoplasmically localized conformation of Ykt6p is important for its sorting by functioning as a conformational sorting epitope. To understand the detailed sorting mechanism of Ykt6p, I conducted studies to crystallize of cytoplasmic form of Ykt6p purified from yeast cells as well as used this protein in Ykt6p / liposome binding assays. My results suggest that the cytoplasmic form of yeast Ykt6p can not mediate direct binding to lipids.