Efficient farnesylation of an extended C-terminal C(x)3X sequence motif expands the scope of the prenylated proteome

Abstract

Protein prenylation is a post-translational modification that has been most commonly associated with enabling protein trafficking to and interaction with cellular membranes. In this process, an isoprenoid group is attached to a cysteine near the C terminus of a substrate protein by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I or II (GGTase-I and GGTase-II). FTase and GGTase-I have long been proposed to specifically recognize a four-amino acid CAAX C-terminal sequence within their substrates. Surprisingly, genetic screening reveals that yeast FTase can modify sequences longer than the canonical CAAX sequence, specifically C(x)₃X sequences with four amino acids downstream of the cysteine. Biochemical and cell-based studies using both peptide and protein substrates reveal that mammalian FTase orthologs can also prenylate C(x)₃X sequences. As the search to identify physiologically relevant C(x)₃X proteins begins, this new prenylation motif nearly doubles the number of proteins within the yeast and human proteomes that can be explored as potential FTase substrates. This work expands our understanding of prenylation's impact within the proteome, establishes the biologically relevant reactivity possible with this new motif, and opens new frontiers in determining the impact of non-canonically prenylated proteins on cell function.

Keywords: Ras protein; enzyme; mass spectrometry (MS); post-translational modification; protein farnesylation; protein isoprenylation; yeast genetics.

Figure 1.

Prenylation pathway recognition and modification of proteins terminating in CAAX sequences. a, protein modification steps observed within the prenylation pathway, including a shunt pathway for proteins undergoing only prenylation without subsequent proteolysis (32). b, structural model of FTase recognition of a CVLS substrate sequence. Recognition of the length of the CVLS tetrapeptide (green) involves coordination of the cysteine side chain thiol to the catalytic zinc ion (orange sphere) and both direct and water-mediated (teal spheres) hydrogen bonding between the peptide C-terminal carboxylate group to FTase residues in both the α (Q167α) and β (H149β) enzyme subunits. Image was generated from Protein Data Bank code 1TN8 using PyMOL (77).

Figure 2.

Phenotypes and isoprenylation status of C(x)₃X motifs identified by yeast-based screening. a, a-factor C(x)₃X variants encoded in CEN LEU2 plasmids and transformed into SM2331 (MATa mfa1 mfa2) were evaluated for their ability to produce a-factor using a spot halo assay; the CGGDD variant was encoded in a high-copy 2μ URA3 plasmid but otherwise treated identically. Strains were spotted onto YPD, cultured for 48 h at 30 °C, and replica-transferred onto a thin lawn of RC757 (MATα sst2-1). Plates were imaged after 16 h of incubation at 30 °C. The same strains were subjected to quantitative mating analyses, which yielded the numerical values indicated below each image, where values are reported as percent relative to control (CVIA). b, Ydj1p C(x)₃X variants encoded in low-copy CEN plasmids were evaluated for their ability to rescue growth of yWS304 (ydj1Δ) at indicated temperatures. Each set of spots represents a 10-fold dilution series prepared from a saturated culture grown in selective media that was spotted onto YPD. Images are representative of data from two separate experiments in which at least two replicates of each strain were evaluated. c, immunoblot of lysates from strains containing the indicated Ydj1p C(x)₃X variant. Farnesylated Ydj1p has increased mobility compared with unmodified Ydj1p. The strains used were yWS304 (WT) and yWS1632 (ram1); RAM1 encodes the FTase β subunit.

Figure 3.

Dansyl-GC(x)₃X peptides can be efficiently farnesylated by mammalian FTase. a, fluorescence-based screening for FTase-catalyzed farnesylation of Dns-GC(x)₃X peptides. b, farnesylation of Dns-GCMIIM (top) and Dns-GCAVGP (bottom) by FTase as monitored by fluorescence enhancement reported in arbitrary units (AU). Red trace, farnesylation reaction; blue trace, control reaction lacking FPP. c, RP-HPLC analysis of FTase-catalyzed farnesylation of Dns-GCMIIM (left) and Dns-GCAVGP (right); substrate and farnesylated product peaks are labeled. Red trace, farnesylation reaction; blue trace, control reaction lacking FPP. d, ESI MS/MS analysis of farnesylated Dns-GCMIIM (left) and Dns-GCAVGP (right); C(Fr) indicates farnesylated cysteine. Reactions were performed and analyzed as described under “Experimental procedures”; tables of fluorescence screening data and ESI MS/MS ion assignments are included in the supporting data.

Figure 4.

C(x)₃X sequence is efficiently farnesylated in the context of a protein substrate. a, LC-MS analysis of farnesylation of an eGFP reporter protein terminating in a C(x)₃X sequence. LC chromatogram of in vitro farnesylation of eGFP-GCAVGP using purified FTase in the absence (panel i) or presence (panel ii) of FPP, with absorbance detected at 555 nm. Negative absorbances are observed due to background fluorescence from eGFP. Peaks A (panel iii) and B (panel iv) have deconvoluted masses of 28,205.1 and 28,408.6 Da, respectively, that differ by 203.5 Da approximately corresponding to farnesyl modification (theoretical mass of farnesyl group: 204 Da). b, in-gel fluorescence scan (top) and Coomassie staining (bottom) of eGFP-GCAVGP subjected to in vitro prenylation using purified FTase in the presence or absence of C15AlkOPP.

Figure 5.

eGFP-KRas-CMIIM is efficiently modified by FTase within a mammalian cell. a, representative images of HEK293 cells transfected with eGFP-KRas-XMIIM or eGFP-KRas-CMII reporter proteins in the absence or presence of tipifarnib (FTI); scale bar, 20 μm. b, scoring of fluorescence patterns observed in HEK293 cells after transfection with eGFP-KRas reporter proteins; an asterisk indicates no cells exhibited membrane-associated fluorescence. Detailed scoring data are provided in Table S3. c, in-gel fluorescence scan (top) and Coomassie staining (bottom) of lysates from HEK293 cells transfected with eGFP-KRas reporter proteins and metabolically labeled with C15AlkOPP followed by conjugation of a TAMRA-N₃ fluorophore. Cells were either non-transfected (HEK293) or transfected with eGFP-KRas-CVIM, eGFP-KRas-CMIIM, or eGFP-KRas-SMIIM reporter proteins in the absence or presence of C15AlkOPP. d, volcano plot for TMT-labeled quantitative proteomic analysis of eGFP-KRas-CMIIM- versus eGFP-KRas-SMIIM-transfected HEK293 cells treated with C15AlkOPP and enriched via biotin-avidin pulldown. A two-sample t test (FDR = 0.05, s0 = 0.5) from three replicates shows that GFP and KRas are statistically enriched in eGFP-KRas-CMIIM transfected cells.