Lovastatin Can be Used to Produce Non-Prenylated ykt6 from PC12 Cells
Bimber, Benjamin N.
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Intracellular trafficking of proteins and lipids is fundamental to normal cell function. Essential to trafficking are SNARE proteins. SNAREs on opposing membranes interact to form SNARE complexes which bring the opposing membranes into close proximity and induce fusion. The SNARE ykt6 is believed to be involved in an uncharacterized transport pathway unique to neurons. Ykt6 exists in both membrane bound and soluble unbound forms. Unlike membrane bound ykt6, soluble ykt6 cannot participate in SNARE complex formation and therefore does not participate in vesicle fusion. If soluble ykt6 is able to revert to a membrane bound form this could also allow ykt6 a degree of targeting flexibility not available to SNAREs which cannot assume a soluble form. It would also represent a novel form of control of SNARE activity. We hypothesize that a conformational change in ykt6 caused by a prenyl moiety results in the inability of soluble ykt6 to form SNARE complexes. In order to test this hypothesis, we will produce and test the SNARE binding ability of non-prenylated ykt6. This study has established a protocol for producing and purifying non-prenylated ykt6 from PC12 cells using the drug lovastatin. We have also tested both gel filtration and GST bead binding assays to analyze SNARE complex formation; however, a successful assay has not been conducted at this time. Future studies will continue work towards an assay to test the SNARE binding ability of non-prenylated ykt6. These experiments will provide tools necessary for the advancement of our understanding of the regulation of ykt6 and neuronal transport in general.