Date of Award

2003

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Robert E. Gundersen

Second Committee Member

Keith W. Hutchison

Third Committee Member

Mary Rumpho-Kennedy

Abstract

Heterotrimeric guanine nucleotide binding proteins (G-proteins) are essential components of a wide variety of eukaryotic cellular signaling pathways. Heterotrimeric G proteins consist of a 40 kDa α-subunit, a 36 kDa β-subunit and a small 8-10 kDa γ- subunit. Acting as molecular switches, G proteins relay molecular information fiom membrane bound receptors to downstream intracellular effectors. Most G-proteins require lipid modification by myristic acid and palmitic acid for proper localization and function. Protein palmitoylation is a post-translational, reversible thioester linkage of palmitic acid (C16:O) to an N- terminal cysteine residue of a substrate protein. Palmitoylation of G-proteins occurs specifically on the a subunit. In the slime mold Dictyostelium discoideum, the transition fiom vegetative growth to multicellular development during starvation has been shown to be dependent upon the G-protein G2. The Gα2 subunit has been shown to be palrnitoylated, in vivo; however, the mechanism by which this modification occurs has proven to be elusive. Recently, Erf2p, a 41 kDa membrane bound protein, has been identified as a Ras palmitoyltransferase in Saccharomyces cerevisiae. Erf2p contains a zinc finger DHHC Cysteine Rich Domain (DHHC-CRD) that has been suggested to be involved in protein-protein or protein-DNA interactions. To determine whether Erf2p homologs exist in Dictyostelium, a BLAST search against the sequenced Dictyostelium genome was performed. A family of twelve Erf2p putative homologs was identified within the genome. To determine whether these putative homologs are expressed during the Dictyostelium life cycle, RT-PCR was performed using primers designed around the internal DHHC sequence found in each of the newly identified open reading frames. Amplified bands of the expected size in each reaction suggest that all twelve sequences are expressed during the life cycle. Real time PCR experiments showed that several of the open reading frames exhibit peak expression at 8 hours of starvation, making these genes candidates to study the palmitoylation of the Gα2 subunit.

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