Purification of a protein palmitoyltransferase that acts on H-Ras protein and on a C-terminal N-Ras peptide.
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.
Liu
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Department of Chemistry, University of Washington, Seattle, Washington 98195-1700, USA.
Dudler
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Gelb
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MH
D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000378:metabolism*; D000818:Animals; D008862:Microsomes, Liver / Q000201:enzymology*; D010171:Palmitoyl Coenzyme A / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D013379:Substrate Specificity; D018631:ras Proteins / Q000378:metabolism*
D000214:Acyl Coenzyme A; D010446:Peptide Fragments; D010171:Palmitoyl Coenzyme A; C040349:S-tetradecanoyl-coenzyme A; D000217:Acyltransferases; C099553:G-protein palmitoyltransferase; D018631:ras Proteins