Cellular palmitoylation and trafficking of lipidated peptides

Jeremiah M. Draper, Zuping Xia, and Charles D. Smith

Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC 29425

Corresponding Author: smithchd{at}musc.edu

Many important signaling proteins require the post-translational 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 the present studies, 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 co-localized with Golgi and plasma membrane markers, whereas, the corresponding non-palmitoylatable peptides accumulate 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.

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