Proteomic identification of palmitoylated proteins

Abstract

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.

FIGURE 1

ABE-purified proteins from yeast membranes. Proteins, detergent-extracted from total yeast membranes, were subjected to the parallel plus- and minus-hydroxylamine ABE protocols. The resulting biotinylated proteins, affinity purified by streptavidin-agarose and eluted through cleavage of protein-biotin links with 1% β-mercaptoethanol, were subjected to SDS-PAGE and silver-staining. Protein bands present exclusively in just the plus-hydroxylamine sample, i.e. the candidate palmitoyl-proteins, are indicated with arrows, while protein species that are present in both samples, i.e. non-specifically purified are indicated by hash marks.

FIGURE 2

Relative plus- and minus-hydroxylamine sample representations for all of the identified proteins. Each of the 1558 proteins identified from the MudPIT analyses of four paired plus- and minus-hydroxylamine samples is plotted by averaged, normalized spectral counts from the plus-hydroxylamine samples (x-coordinate) and from the minus-hydroxylamine samples (y-coordinate). At right, the indicated portion of the plot is expanded. Known palmitoyl-proteins are indicated in black. The encircled area includes the new candidate palmitoyl-proteins co-clustering with the known palmitoyl-proteins.

FIGURE 3

Yeast proteins, from the 70 top-ranking identified by the proteomic analysis, with confirmed palmitoylation. Proteins are grouped by likely palmitoylation sequence features. The 12 detected proteins known at the outset of this work to be palmitoylated, are indicated by asterisks, while the three known palmitoyl-proteins that were not detected from within the top 70 grouping, are listed at the bottom. Our analysis detected palmitoylation for many yeast SNARE proteins and for many amino acid permeases (AAPs). Proteins lacking strong independent confirmation of palmitoylation are indicated by question marks. Four of the palmitoyl-proteins, newly-identified by this analysis, have also been demonstrated to be palmitoylated in recent published reports, these are Gpa2, Lcb4, Tlg1, and Syn8 (–22).

FIGURE 4

Yeast SNARE proteins detected and not detected by the proteomic analysis. For the 17 yeast SNARE proteins having typical single-TMD architecture, the sequence surrounding the TMD is shown. Cysteines predicted to map near the membrane-cytoplasm interface are indicated. C termini are indicated with dots.