Date of Award

5-2007

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Robert E. Gundersen

Second Committee Member

Dorothy E. Croall

Third Committee Member

Gregory D. Mayer

Abstract

Guanine nucleotide binding proteins (G proteins) are crucial in a broad range of eukaryotic signaling pathways. They function by activating downstream effectors in response to receptor-ligand binding. Most G proteins require a post-translational lipid modification of myristic and/or palmitic acid for correct cellular localization and function. In the soil amobae Dictyostelium discoideum the transition from unicellular to multicellular development is induced by starvation. This transition is dependent upon palmitoylation of G proteins in several signaling pathways. A mechanism by which G proteins are palmitoylated has recently been defined in S. cerevisiae. This Ras palmitoyltransferase gene contains a zinc-finger-like DHHC cysteine rich domain (16). BLAST analysis of the yeast palmitoyltransferase gene sequence identified 14 putative palmitoyltransferases in the Dictyostelium genome, all of which contain a DHHC zinc finger motif similar to that of the identified S. cerevisiae gene sequence. These genes have been termed PAZ1-14 (putative acyltransferase with a zinc finger). Since initiation and progression of the Dictyostelium lifecycle is dependent on a protein that requires palmitoylation, it is pertinent to examine the expression levels of the 14 identified PAZ genes during the course of the 24-hour lifecycle. Using quantitative Reverse Transcriptase Polymerase Chain Reaction (qRTPCR), the expression pattern of most of the PAZ genes was determined. The results revealed that each of the PAZ genes displays a unique pattern of expression, suggesting that each serves a specialized role in the lifecycle of Dictyostelium. Experiments focusing on PAZ5 elucidated several characteristics of the gene. While knocking out PAZ5 did not have any effect on the growth rate of Dictyostelium, overexpressing PAZ5 resulted in a slower growth rate, suggesting that the pathways regulated by the enzymatic activity of PAZ5 are most likely involved in the suppression of replication. The phenotype of Dictyostelium is abnormal both when PAZ5 is knocked out, and when PAZ5 is overexpressed, indicating a crucial role for PAZ5 in correct developmental assembly of different cellular structures.

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