(A) Electrophoretic analysis of purified −hydroxylamine (CON) and +hydroxylamine (EXP) samples. One percent of total purified samples were subjected to SDS-PAGE and silver staining. EXP sample-specific proteins are indicated by arrows.
(B) Graphical depiction of MS analysis. For each identified protein, averaged and normalized EXP sample spectral counts from four wild-type samples (x axis) is plotted against averaged, normalized CON sample spectral counts (y axis). Shown at right is an expanded view of the indicated portion. Known palmitoyl proteins are indicated in red; the new candidate palmitoyl proteins are blue. Proteins with substantial representations from both the EXP and CON samples are gray. Note that 3 of the 15 known palmitoyl proteins fail to cluster with other known palmitoyl proteins, being detected either by low spectral count numbers (Ykt6) or from both EXP and CON samples (Hem14 and Tub1). See Table S1 for supporting MS/MS data.

Palmitoyl proteins, both new and known, detected by the MS analysis are classed by presence or absence of predicted TMDs and by positioning of likely palmitoyl-accepting cysteines. Known palmitoyl proteins are shown in brown; new palmitoyl proteins are shown in black. For each of the listed proteins, Table S2 provides a full summary of the evidence supporting palmitoylation. Proteins lacking strong independent confirmation of palmitoylation are shown in green. Four proteins for which palmitoylation has been confirmed in recent published reports (Harashima and Heitman, 2005; Kihara et al., 2005; Valdez-Taubas and Pelham, 2005) are shown in red. At right, TMD-proximal sequences are shown for the indicated proteins.

(A) Consensus elements for the 11 amino acid permeases (AAPs) with C-terminal cysteines. The five AAPs identified as palmitoylated by the proteomic analysis are indicated with asterisks.
(B) AAP C-terminal Cys is required for palmitoylation. For the indicated AAPs, palmitoylation of both the wild-type AAP (wt) and the C-terminal Cys-to-Ser mutant version (−FWS) was assessed using a scaled-down version of the acyl-biotinyl exchange (ABE) protocol (Supplemental Experimental Procedures). To facilitate this analysis, AAPs were N-terminally FLAG/HA epitope tagged and overexpressed from the GAL1 promoter (2 hr expression period). Following ABE, AAPs were anti-FLAG immunoprecipitated and then blotted for biotinylation (α-biotin; indicative of palmitoylation) or overall recovery (α-HA).
(C) Membrane tethering sequences of yeast Rho proteins. The C-terminal 13 amino acid residues of the six yeast Rho proteins are shown. CaaX prenylation motifs and polybasic domains are red, and likely palmitoyl-accepting cysteines are highlighted in black.
(D) N-terminal Rho3 palmitoylation requires prior C-terminal prenylation. Cells expressing wild-type or the two indicated mutant Rho3 proteins from the GAL1 promoter (2 hr expression period) were labeled with [³H]palmitic acid. Immunoprecipitated Rho3 was analyzed for both label incorporation (top panel) and Rho3 protein recovery (bottom panel). The 4× HA/FLAG epitope tag used for this analysis (for immunoprecipitation and for Western detection) was inserted internally within the RHO3 ORF, between codons Ala217 and Thr218 (14 codons from the RHO3 stop codon), to avoid disrupting N- and C-terminal lipidation sites.

For the 30 top-ranking palmitoyl proteins, EXP sample representations (based on normalized spectral counts; see Table S3 for supporting MS/MS data) from the different mutant strains were compared to averaged EXP representations derived from the four wild-type MS/MS runs. The relevant genotypes of the different mutant strains are indicated at left: wild-type alleles (+), deletion alleles (Δ), or depletion alleles (red downward arrow). Proteins with 20-fold or greater mutant strain underrepresentations are depicted as red, with intermediate levels of underrepresentation converted to intermediate shadings of red as described (Supplemental Experimental Procedures). Similarly, proteins with mutant sample overrepresentations are depicted by shadings of green. (Subtle green shadings are faintly apparent for only a few boxes within this matrix.) For strains utilizing GAL1-driven ERF2 or SWF1 depletion alleles, the depletion time period (period of growth on glucose medium) is indicated at right. The row numbers in yellow at left allow the results from individual strains to be referenced from the text. An EXP sample from the isogenic wild-type parent strain was analyzed as a control (row 11). Note that some of the profiled strains harbor the akr1-suppressing lys2Δ::YCK2(CCIIS) allele (as indicated). The substantial Yck2 palmitoylation that is found to persist in some of the profiled akr1Δ strains (e.g., row 7) likely reflects ongoing Akr1-independent palmitoylation of the Yck2(CCIIS) mutant.

For the indicated proteins, palmitoylation in the wild-type strain context (+) was compared with that in the indicated DHHC gene-deletion strain context (Δ): akr1Δ (A), erf2Δ (B), or swf1Δ (C). Palmitoylation was assessed by the small-scale ABE protocol as described for Figure 3B. The tested proteins were expressed from the GAL1 promoter and were either N- or C-terminally tagged with the dual HA/FLAG epitope tag (see Table S2). For (A) and (B), putative Akr1 and Erf2-Shr5 substrates are listed and sequences surrounding likely palmitoyl-accepting cysteines are shown. Note that Sna4 and Meh1 are unique among the putative Akr1 substrates in that Sna4 has two predicted TMDs and Meh1 is predicted to be heterolipidated: myristoylated in addition to palmitoylated. The asterisk marks a nonspecific band that crossreacts with the anti-biotin IgG.

Palmitoylation of the indicated AAPs was assessed by the small-scale ABE protocol as described for Figure 3B.
(A) Deconvolution of the DHHC protein requirement for Tat1 palmitoylation.
(B) Pfa4-dependent palmitoylation for the Phe-Trp-Cys-containing AAPs.