Abstract
Mammalian H-Ras and N-Ras are GTP-binding proteins that must be post-translationally lipidated to function as molecular switches in signal transduction cascades controlling cell growth and differentiation. These proteins contain a C-terminal farnesyl-cysteine alpha-methyl ester and palmitoyl groups attached to nearby cysteines. Data is presented showing that rat liver microsomes contain an enzyme that transfers the palmitoyl group from palmitoyl-coenzyme A to cysteine residues of H-Ras protein and of a synthetic peptide having the structure of the C terminus of N-Ras. This protein palmitoyltransferase (PPT) was solubilized from membranes and purified 10,500-fold to apparent homogeneity with an overall yield of 10%. On an SDS gel, PPT appears as two proteins of molecular masses of approximately 30 and approximately 33 kDa. If the palmitoylation sites of the N-Ras peptide (the non-farnesylated cysteine) or H-Ras protein (cysteines 181 and 184) are changed to serine, palmitoylation by PPT does not occur. Non-farnesylated H-Ras produced in bacteria as well as in vitro farnesylated bacterial H-Ras are not substrates for PPT nor is the non-farnesylated, methylated N-Ras peptide. These results suggest, but do not prove, that farnesylation and possibly C-terminal methylation are prerequisites for Ras palmitoylation. PPT shows a large preference for palmitoyl-coenzyme A over myristoyl-coenzyme as the acyl donor. Values of Km for palmitoyl-CoA and H-Ras are 4.3 +/- 1.2 and 0.8 +/- 0.3 microM, respectively. PPT is the first protein palmitoyltransferase to be purified, and the availability of pure enzyme should contribute to our understanding of the function and regulation of Ras palmitoylation in cells.
Highlights
§ Recipient of postdoctoral fellowships from the Swiss National Science Foundation and the Schweizerische Stiftung fur Medizinisch-Biologische Stipendien
Enzymatic Palmitoylation of (N-Ras-16mer)—Based on the previous demonstration of N-Ras palmitoylation in a cell-free preparation made from rat liver [30], a synthetic peptide having the C-terminal 16 residues of human N-Ras and a Cterminal S-farnesyl cysteine methyl ester (N-Ras-16-mer) was first used to detect and characterize a protein palmitoyltransferase (PPT) activity present in rat liver microsomes
Further evidence that the RF 0.44 spot is palmitoylated N-Ras-16-mer is the following: 1) when this spot was eluted from the TLC plate, de-palmitoylated and demethylated with KOH, and radioacetylated with [3H]acetic anhydride, a single peak of radioactivity was seen by HPLC analysis that has the same retention time as authentic N-Ras-16-mer treated in an identical manner
Summary
This spot was not seen if the assay was carried out with the peptide in which the cysteine residue of N-Ras-16-mer that is not farnesylated (the putative palmitoyl acceptor) contains an S-t-butylthio group instead of SH (Fig. 1A). Enzymatic Palmitoylation of H-Ras—A second assay for PPT was developed using fully processed H-Ras produced in baculovirus-infected insect cells [28] that was chemically depalmitoylated with a neutral solution of NH2OH under conditions which do not lead to C-terminal demethylation (“Experimental Procedures”).
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