Interactions of Human VPS45 with Partner SNARE Proteins

Huaman, Kimberly

Student Work

Interactions of Human VPS45 with Partner SNARE Proteins

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Intracellular trafficking is a fundamental cellular process regulating the movement of materials between organelles and to the plasma membrane. The steps of an intracellular trafficking pathway include: budding of the vesicle from the donor membrane, vesicle transport to the target membrane, vesicle tethering and docking at the target membrane, and, finally, fusion between the vesicle and target membranes. This process is highly regulated and involves the coordination of many proteins such as SNAREs and Sec1/Munc18 (SM) proteins. Among the SNAREs, Syntaxin 16 (Stx16) drives SNARE complex assembly at the endosome. VPS45, an endosomal SM protein, is proposed to interact with Stx16 and regulate membrane fusion. Interestingly, VPS45 variants have been identified in patients with Severe Congenital Neutropenia (SCN). This study used size exclusion chromatography (SEC) to investigate the binding of VPS45-His6 to His6-Stx16, other endosomal SNAREs, and the endosomal SNARE complex. Unfortunately, SNARE complexes did not form, in the presence or absence of VPS45-His6 under the listed conditions. SEC results revealed that wild-type VPS45-His6 interacts with His6-Stx16 but not other endosomal SNAREs. In future studies, these experiments will be repeated under conditions closer to a physiological state. Alternative assays, such as native gel electrophoresis or pulldowns, will also be used. Finally, variants of VPS45 present in SCN will be compared to wild-type VPS45 in these assays to determine their effect on SNARE assembly.

This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

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