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Originally published In Press as doi:10.1194/jlr.M700179-JLR200 on May 24, 2007

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Journal of Lipid Research, Vol. 48, 1873-1884, August 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Cellular palmitoylation and trafficking of lipidated peptides

Jeremiah M. Draper*, Zuping Xia{dagger} and Charles D. Smith1,*,{dagger}

* Department of Pharmacology, Pennsylvania State University College of Medicine, Hershey, PA
{dagger} Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC

Published, JLR Papers in Press, May 24, 2007.

1 To whom correspondence should be addressed. e-mail: smithchd{at}musc.edu

Many important signaling proteins require the posttranslational addition of fatty acid chains for their proper subcellular localization and function. One such modification is the addition of palmitoyl moieties by enzymes known as palmitoyl acyltransferases (PATs). Substrates for PATs include C-terminally farnesylated proteins, such as H- and N-Ras, as well as N-terminally myristoylated proteins, such as many Src-related tyrosine kinases. The molecular and biochemical characterization of PATs has been hindered by difficulties in developing effective methods for the analysis of PAT activity. In this study, we describe the use of cell-permeable, fluorescently labeled lipidated peptides that mimic the PAT recognition domains of farnesylated and myristoylated proteins. These PAT substrate mimetics are accumulated by SKOV3 cells in a saturable and time-dependent manner. Although both peptides are rapidly palmitoylated, the SKOV3 cells have a greater capacity to palmitoylate the myristoylated peptide than the farnesylated peptide. Confocal microscopy indicated that the palmitoylated peptides colocalized with Golgi and plasma membrane markers, whereas the corresponding nonpalmitoylatable peptides accumulated in the Golgi but did not traffic to the plasma membrane. Overall, these studies indicate that the lipidated peptides provide useful cellular probes for quantitative and compartmentalization studies of protein palmitoylation in intact cells.

Supplementary key words palmitoyl acyltransferase • Ras • lipidation • subcellular trafficking

Abbreviations: AUC, area under the curve; LDH, lactate dehydrogenase; NBD, 7-nitro-2-1,3-benzoxadiazol-4-yl; PAT, palmitoyl acyltransferase; WGA, wheat germ agglutinin


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Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.