Identifying New Substrates for PFA3: A Protein Fatty Acyltransferase

Audrey Musi Ye, Maurine E. Linder, Ph.D., Department of Cell Biology and Physiology, Washington University School of Medicine

Proteins are essential to cells. Studying their cellular functions and regulation, especially their methods of localization, are vital to understanding their links to illnesses such as cancer and neuropsychiatric diseases.  Some proteins are modified by lipid attachment to their polypeptide chain.  One such modification is N-myristoylation in which myristate, a 14-carbon fatty acid is attached to an N-terminal glycine residue.  Another is S-palmitoylation in which palmitate, a 16-carbon fatty acid, is added to cysteine residues through an S-thioester bond; this is a reversible reaction.  These additions increase the hydrophobicity of proteins and thus help proteins with membrane affinity and their subcellular trafficking between different organelle membranes or within membranes. Both are enzymatic reactions; N-myristoylation is mediated by N-myristoyltransferase (NMT) while S-palmitoylation is mediated by protein acyltransferases (PATs), but the exact enzymology of the latter mechanism is not completely clear.  Recent findings in Saccharomyces cerevisiae [yeast] have identified two PATs and their substrates.   Both PATs share an Asp-His-His/Tyr-Cys sequence in a cysteine rich domain (DHHC-CRD) that is needed for palmitoylation indicating that DHHC-CRD might be a conserved domain for PATs.  This domain is also found in five other yeast proteins, which make theses proteins an interest for study as potential PATs.  

A particular protein of interest is Pfa3p, localized in the yeast vacuole.  Based on previous work in the Linder lab, a candidate substrate Vac8p, which is both myristoylated and palmitoylated, was found.  If other substrates could be identified, they could be compared to Vac8p to further study characteristics such as the specificity of PATs.  Two genes of interest from yeast were selected to test for as substrates: YGL108C and MEH1.  Both have domains that are correct for myristoylation and palmitoylation, and MEH1 is also found on the yeast vacuole. 

To study these two genes, they were first isolated from genomic DNA using PCR and inserted into TOPO vectors.  A MYC tag was added on the C-terminus, and restriction sites were also added.  It was then digested and ligated into pQE60 vectors and screened by bacteria colony PCR.  A positive clone of MEH1 was transformed into JM109 cells along with NMT to simulate myristoylation conditions.  It was induced with 0.3 mM IPTG to lift repressor off the promoter to start protein expression, and a western blot was used to test for protein expression.  No expression of the MEH1 protein was seen, and temperatures of growth and concentration of IPTG induction were changed to stimulate protein expression.  It was later identified that the clone was a false positive, and to continue the study, a new vector will be constructed for enhanced expression.