Study On The Mechanism Of Trafficking And Targeting Of Protein Related To Vesicle Transport

Cell secretion contain a complex series of processes, and the SNARE complex is oneof the key proteins. It is generally believed that the interaction of the SNARE proteinsprovides the driving force for vesicle fusion. Syntaxin1A is mainly located on the plasmamembrane. Syntaxin plays a key role in intracellular membrane fusion in eukaryotic cells.The function of Syntaxin relies on its proper trafficking to and targeting at the targetmembrane. The conservation of the N-terminal Habc domain implies that it is importantfor membrane syntaxin function.The mechanisms of targeting of STX1A to its target sites are still poorly understood.To this end, we have analyzed the trafficking of Syntaxin1A in INS-1and CHO cells byusing Syntaxin1A （Stx1A） mutants. We constructed a series of Syntaxin1A mutants,which have pHluorin tagged on their C-terminals, to study them in the two types of cellson the trafficking.We found that the trans-membrane domain plus several positive-charged amino acidsof Syntaxin1A is the minimal domain required for the membrane delivery.Interestingly,we found that SNARE motif-exposed Syntaxin1A mutants retained in endoplasmicreticulum （ER） and failed to transport to the cell surface in the absence of SNAP-25. Itsuggested that that the exposure of the SNARE-motif of Syntaxin1A causes its retentionin ER and the interaction with SNAP-25helps its escape from ER. Our data propose twokey roles for the H_abc domain: to protect nonspecific interaction by masking theSNARE-motif and to participate in the clustering of Syntaxin1A to the fusion sites in theplasma membrane. Moreover, partly truncated H_abc domain mutants still transport to thecell surface, even in the absence of SNAP-25. But they could not target at the functionalsites （clusters） as wild type Stx1A. Moreover, Munc18-1is not essential in the clusterdistribution of Stx1A.