Date of Award

Fall 12-18-2020

Level of Access

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Robert E. Gundersen

Second Committee Member

Julie A. Gosse

Third Committee Member

Joshua B. Kelley

Abstract

Protein S-palmitoylation plays a crucial role in many biological systems. S-palmitoylation involves the post-translational attachment of palmitate to a cysteine residue through a reversible thioester linkage. S-Palmitoylation is used to modify both integral and membrane proteins, many of which are involved in intracellular trafficking, membrane localization, and signal transduction pathways. Intracellular palmitoylation is mediated by a family of protein acyltransferases (PATs). PAT mutations are associated with neurological diseases and cancer progression. Proteins in the PAT family are defined by the presence of a 51-amino acid cysteine-rich domain (CRD), which contains a highly conserved aspartate-histidine-histidine-cysteine (DHHC) motif. The structure and function of DHHC proteins are still under investigation due to the difficulty in purifying eukaryotic multi-pass transmembrane proteins.

In the model eukaryotic organism Dictyostelium discoideum, fourteen DHHC proteins have been discovered, labeled PAZ1-14. Previous work has investigated the localization of these proteins during growth and differentiation, with attention paid towards their effect on palmitoylation of the Gα2 subunit of the G protein. The PAZ5 protein was chosen for further investigation as cells transformed with a knockout PAZ5 presented a unique malformed phenotype in the fruiting body stage of the D. discoideum lifecycle. The fruiting body would collapse as fewer cells differentiated into stalk cells; the cell lines would return to normal functioning after rescue with the wildtype. It was also previously shown that mutations to the DHHC motif of PAZ5 induced the same malformed phenotype as the knockout, indicating a crucial role for PAZ5 in D. discoideum development.

Using site-directed mutagenesis, seventeen independent mutations to PAZ5 were created, both in the DHHC-CRD and other conserved motifs. These were ligated into a fluorescent-tagged extrachromosomal expression plasmid (pTX-GFP) and transformed into D. discoideum. They were analyzed for fluorescent localization and the previously seen fruiting body malformations. Unforeseen low transformation efficiency prevented the full use of all seventeen mutants. The mutants that were able to be analyzed produced a phenotype dissimilar to both the wildtype and knockout strains; moreover, fluorescent localization of PAZ5 was unable to be discerned. It was determined that the plasmid experienced a loss of function, though this was not apparent until after the results were collected. Consequently, it is unknown to what extent the observations are experimentally valid. The work can be salvaged by re-ligating the mutant PAZ5 proteins into a new fluorescent expression plasmid. Further investigation into the DHHC-CRD of PAZ5 would provide an important eukaryotic analog for PAT research.

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