Purification of a Protein Palmitoyltransferase that Acts on H-Ras Protein and on a C-terminal N-Ras Peptide

doi:10.1074/jbc.271.38.23269

Purification of a Protein Palmitoyltransferase that Acts on H-Ras Protein and on a C-terminal N-Ras Peptide*

From the Departments of Chemistry and Biochemistry, University of Washington, Seattle, Washington 98195-1700

^¶To whom correspondence should be addressed:
Depts. of Chemistry and Biochemistry, Box 351700, University of Washington, Seattle, WA 98195-1700
. Tel.: 206-543-7142; Fax: 206-685-8665; E-mail: gelb{at}chem.washington.edu.

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 α-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 ≈30 and ≈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 K_m for palmitoyl-CoA and H-Ras are 4.3 ± 1.2 and 0.8 ± 0.3 μ, 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.

Footnotes

↵* This work was supported in part by Grant CA52874 and Research Career Development Award GM562 from the National Institutes of Health (to M. H. G.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵^‡Recipient of a George H. Hitchings, Jr. Graduate Student Fellowship.
↵^§Recipient of postdoctoral fellowships from the Swiss National Science Foundation and the Schweizerische Stiftung für Medizinisch-Biologische Stipendien.
↵¹ The abbreviations used are:

N-Ras-16-mer

LNSSDDGTQGCMGLP-(S-farnesyl-cysteine-α-carboxyl methyl ester)

BSA

bovine serum albumin

CoA

coenzyme A

PPT

protein palmitoyltransferase from rat liver that transfers the palmitoyl group of palmitoyl-CoA to cysteines near the C termini of H-Ras protein or present on N-Ras-16-mer

PAGE

polyacrylamide gel electrophoresis

Pipes

1,4-piperazinediethanesulfonic acid.
- Received January 29, 1996.
- Revision received July 10, 1996.