Date of Award

12-2010

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Robert E. Gundersen

Second Committee Member

Keith W. Hutchison

Third Committee Member

Sharon Ashworth

Abstract

The ability of many proteins to signal appropriately is dependent upon the post-translational attachment or cycling of a palmitic acid moiety. Studies in Saccharomyces cerevisiae have revealed a family of protein acyltransferases (PATs) containing a conserved cysteine-rich domain (CRD) with a key asp-his-his-cys (DHHC) sequence directly involved in palmitoylation of target proteins including Ras (34). Studies attempting to reveal areas of activity have shown DHHC-CRD proteins localized to the ER, Golgi, and plasma membranes (26) and have shown the possibility for substrate overlap between PATs (35). To determine the role of the DHHC-CRD family of proteins in signaling we have begun cloning of the 14 genes and characterization of the resulting proteins found in Dictyostelium discoideum. These have been investigated for localization, function, and their effect on localization of the known palmitoylated Gα2 subunit. A PAZ4-GFP fusion protein was determined to localize to the Golgi, PAZ10-GFP to the ER, and PAZ8-GFP to both the Golgi and ER. Cell lines deficient in PAZ5 present a phenotype of slowed development and malformed fruiting bodies that can be rescued by overexpression of a PAZ5 cDNA. Mutations introduced into the DHHC-CRD of the PAZ5 cDNA construct abohshed its abihty to rescue the phenotype suggesting a key role for the DHHC-CRD. This work provides a repeatable foundation for further investigation into this important family of proteins.

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