EXPANDING FARNESYLTRANSFERASE SPECIFICITY BEYOND THE CANONICAL CAAX CONSENSUS SEQUENCE

Protein lipidation is a post-translational modification (PTM) that commonly occurs to proteins controlling eukaryotic cell growth, differentiation, and morphology. Farnesyltransferase (FTase) covalently attaches a farnesyl lipid to a conserved cysteine near the C-terminus of these proteins, which increases protein hydrophobicity and affinity towards cellular membranes. Much of what is known about farnesylation was pioneered with Ras-based studies because hyperactive Ras signaling is implicated in roughly 30% of all human cancers. From these early studies, the FTase recognition sequence was defined as a C-terminal Cysteine – aliphatic – aliphatic - variable (CaaX) motif. However, growing evidence including the work presented in this dissertation indicates that there are many more FTase targets that do not adhere to the canonical CaaX motif. Furthermore, newly-revealed FTase substrates in Saccharomyces cerevisiae, exemplified by the yeast HSP40 chaperone Ydj1, are subject only to farnesylation unlike canonical CaaX proteins such as Ras that undergo multi-step PTM (i.e. farnesylation, proteolysis, carboxylmethylation). These observations challenge the generally accepted specificity of FTase and has led to the hypothesis that additional FTase targets with non-canonical CXXX motifs are likely present in eukaryotes. Because biochemical and proteomic methods are impractical for evaluating the specificity of FTase toward all 8000 possible CXXX permutations, we developed yeast screening methods utilizing Ydj1-CXXX as a robust reporter of FTase activity. This approach has provided the first comprehensive profile for yeast FTase specificity in vivo confirming a broader range of targets for this enzyme than previously reported. FTase and its targets are well-conserved amongst eukaryotes, and our findings raise a strong possibility that many additional FTase targets in humans are likely prevalent. The work presented in this dissertation provides a vigorous approach to assess FTase specificity, which can be used for better predictions of the prenylome and potential impacts of therapies utilizing FTase inhibitors for conditions like cancer, progeria, and parasitic diseases.