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PhD Gradute: Tran Thi Ton Hoai
Thesis Title: Interaction of Scribble with Zonula Occludens and Intermediate Filament Proteins.
SNAREs (soluble N-ethylamaleimide sensitive factor attachment protein receptor) are key regulators of the last stage of vesicle docking and subsequent fusion in diverse intracellular membrane transport events. Since the function of SNAREs is regulated primarily by their localization, it is important to understand their targeting mechanisms. VAMP4 (vesicle-associated membrane protein 4) is a SNARE located mainly in the trans-Golgi network (TGN). It functions in membrane traffic from the sorting and recycling endosomes to the TGN, but its trafficking itinerary is unknown. The N-terminal domain preceding the SNARE motif of VAMP4 contains an autonomous targeting signal for the TGN, which resides in a region consisting of a double-Leu motif followed by two acidic clusters. For detailed study of the role(s) of this region as the targeting signals of VAMP4, Ala-mutagenesis screening was performed. Immunofluorescence study showed that the double-Leu motif and an acidic cluster play essential roles in mediating efficient TGN targeting.
An antibody internalization assay using C-terminally EGFP-tagged VAMP4 also showed that VAMP4-EGFP cycles between the PM and the TGN and that its N-terminal domain may participate in regulating this recycling. Detailed time-course analysis of anti-GFP antibody transport to the TGN as well as pharmacological and thermal perturbation experiments suggest that VAMP4-EGFP is endocytosed by clathrin-dependent pathways. It is then transported to the TGN via the sorting and recycling
endosomes, but not the late endosome. The double-Leu motif of the TGN-targeting signal is important for internalization, whereas the acidic cluster is crucial for endosome-to-TGN transport. Site-directed mutagenesis in VAMP4-EGFP showed that the negative charge and steric size of the phosphorylated Ser-30, which is sandwiched between the two acidic clusters, are important for the TGN-to-PM transport of VAMP4-EGFP. These results suggest that the TGN-targeting signal of VAMP4 mediates the efficient cycling of VAMP4 between the TGN and the PM, thus conferring steady-state enrichment of VAMP4 at the TGN