Type of Document Dissertation Author Liu, Ke URN etd-06282007-185913 Title Trafficking and functional analysis of yeast Drs2 Degree PhD Department Biological Sciences Advisory Committee
Advisor Name Title Kendal Broadie Committee Chair Anne K Kenworthy Committee Member Katherine L Friedman Committee Member Susan R Wente Committee Member Todd R Graham Committee Member Keywords
- protein trafficking
- phospholipid translocase
- clathrin-coated vesicle
Date of Defense 2007-04-04 Availability unrestricted AbstractDrs2p, a phospholipid translocase required for generation of membrane asymmetry, plays a role in the clathrin-coated vesicle formation from the trans-Golgi network (TGN) and endosomal membranes. The mechanism for coupling Drs2p to specific transport pathways requires its appropriate localization. To maintain its steady-state residence at the TGN, Drs2p primarily cycles between the TGN and the early endosome, although it occasionally traffics to the plasma membrane where it is rapidly endocytosed and efficiently retrieved to the TGN from early endosomes.
Endocytosis of Drs2p is mediated by multiple signals, including two NPFXD motifs near the C-terminus and PEST-like sequences near the N-terminus that likely mediate ubiquitin (Ub)-dependent endocytosis. The NPFXD motifs are specifically recognized by Sla1p, part of an endocytic coat/adaptor complex with clathrin, Pan1p, End3p, and Sla2p. When both ubiquitin- and NPFXD-dependent endocytic mechanisms are abrogated, Drs2p accumulates on the plasma membrane. Surprisingly, the pan1-20 temperature-sensitive mutant is constitutively defective for Ub-dependent endocytosis but is not defective for NPFXD-dependent endocytosis at the permissive growth temperature. To sustain viability of pan1-20, Drs2p must be internalized through the NPFXD/Sla1p pathway. Thus, Drs2p is an essential endocytic cargo for the NPFXD/Sla1p system in cells compromised for Ub-dependent endocytosis. These discoveries provide a novel example for the critical role of endocytosis in retrieving Golgi proteins that escape to the plasma membrane.
The clathrin adaptor AP-1 interacts with Drs2p and disruption of AP-1 dramatically increases the rate of Drs2p transport to the plasma membrane. Retrieval of Drs2p back to the TGN seems to be unaffected in AP-1 mutants, since the steady-state localization is not perturbed. Therefore, Drs2p depends on AP-1 for anterograde transport from the TGN to the endosome, but not for endosome to TGN retrograde transport. Importantly, Drs2p is required for AP-1 function, but does not appear to contribute significantly to the GGA/clathrin pathway. AP-1 and clathrin are recruited to TGN and endosomal membranes in the absence of Drs2p but fail to support transport of AP-1/clathrin-dependent cargo. Based on these observations, we propose that Drs2p plays an essential role in budding vesicles by pumping phospholipid to the cytosolic leaflet to generate positive membrane curvature that is captured by AP-1/clathrin coats. Drs2p actually embarks within these AP-1/clathrin-coated vesicles for delivery to the early endosome, but uses an AP-1 independent pathway for retrieval back to the TGN.
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