Protein Isoprenylation in Yeast Targets COOH-Terminal Sequences Not Adhering to the CaaX Consensus

Abstract

Protein isoprenylation targets a subset of COOH-terminal Cxxx tetrapeptide sequences that has been operationally defined as a CaaX motif. The specificity of the farnesyl transferase toward each of the possible 8000 combinations of Cxxx sequences, however, remains largely unresolved. In part, it has been difficult to consolidate results stemming from in vitro and in silico approaches that yield a wider array of prenylatable sequences relative to those known in vivo We have investigated whether this disconnect results from the multistep complexity of post-translational modification that occurs in vivo to CaaX proteins. For example, the Ras GTPases undergo isoprenylation followed by additional proteolysis and carboxymethylation events at the COOH-terminus. By contrast, Saccharomyces cerevisiae Hsp40 Ydj1p is isoprenylated but not subject to additional modification. In fact, additional modifications are detrimental to Ydj1p activity in vivo We have taken advantage of the properties of Ydj1p and a Ydj1p-dependent growth assay to identify sequences that permit Ydj1p isoprenylation in vivo while simultaneously selecting against nonprenylatable and more extensively modified sequences. The recovered sequences are largely nonoverlapping with those previously identified using an in vivo Ras-based yeast reporter. Moreover, most of the sequences are not readily predicted as isoprenylation targets by existing prediction algorithms. Our results reveal that the yeast CaaX-type prenyltransferases can utilize a range of sequence combinations that extend beyond the traditional constraints for CaaX proteins, which implies that more proteins may be isoprenylated than previously considered.

Keywords: CaaX; Hsp40; farnesyl transferase; isoprenylation; thermotolerance.

Figure 1

Site-directed mutation of the Ydj1p Cxxx motif reveals flexibility in sequence requirements for functional levels of isoprenylation. (A) The Cxxx motif directs protein isoprenylation. Both farnesyl (C15) and geranylgeranyl (C20) can be added to Cxxx proteins; only C15 addition is shown for clarity. The isoprenylated species is either the endpoint modification (e.g., Ydj1p; shunted proteins) or an intermediate that is additionally modified by proteolysis and carboxymethylation (e.g., K-Ras4b; traditional CaaX proteins). Not shown are more extensive modifications that can also occur, such as palmitoylation (e.g., H- and N-Ras) or distal proteolysis (e.g., lamin A; yeast a-factor). (B) WebLogo frequency analysis of the last seven amino acids associated with 14 Ydj1p homologs retrieved from the Homologene Database (http://www.ncbi.nlm.nih.gov/homologene). Color scheme is as described in Materials and Materials and Methods. See Figure S1 for specific sequence details. (C) Ydj1p mutants were evaluated for their ability to support high-temperature yeast growth. yWS304 yeast (ydj1∆) expressing the indicated plasmid-encoded Ydj1p mutant were cultured in selective SC-uracil media and pinned as 10-fold serial dilutions onto nonselective YPD; the leftmost spot in each panel is undiluted. Plates were incubated at the indicated temperature as described in Materials and Methods. (D) The Ydj1p mutants indicated in C were expressed in yWS304 (ydj1∆) and cell lysates evaluated by anti-Ydj1p immunoblot. The specific plasmids used for Figure 1 are listed in Table S2. Unmodified Ydj1p (open triangle) migrates at a larger apparent kDa than prenylated Ydj1p (solid triangle).

Figure 2

Effect of temperature and time on yeast thermotolerance. (A) Yeast expressing the indicated Ydj1p variants were cultured to saturation in selective media, diluted into YPD, spotted using a multichannel pipettor onto YPD solid media, and plates incubated at indicated temperatures and times. A 1:20 dilution was the source for spots incubated at 25°, and a 1:2 dilution was used for spots incubated at other temperatures. (B) Yeast were cultured and processed as described for A, using the 40° condition with the following alterations: the 1:2 dilution was into YPD, incubation at 40° was for 72 hr followed by recovery at 25° for the indicated times. (C) Flow diagram of screen used to identify thermotolerant yeast expressing Ydj1p Cxxx variants in either the ydj1∆ or ydj1∆ ste14∆ background. (D) Examples of thermotolerance profiles observed and thermotolerance (T) scores assigned when expressed in ydj1∆ background; the controls and panel of mutants are replicated in Figure S2. Yeast thermotolerance assays were performed as described for Figure 1. (E) The thermotolerance profiles of all recovered mutants were scored relative to yeast expressing Ydj1p with a SASQ, CVIA, or CASQ motif, where the controls were assigned scores of 1, 3, and 5, respectively. The mutants were then binned according to their scores; see Materials and Methods for details on binning.

Figure 3

Isoprenylation and cleavage properties of Ydj1p Cxxx mutants identified through thermotolerance screening. (A and B) Yeast expressing the indicated Ydj1p mutants were evaluated by a gel-shift assay as described in Figure 1C. The sequences evaluated were either randomly identified from (A) the CASQ-like and CASQ-like (weak) groups or (B) reflect the combined set of sequences presenting with CVIA-like and CVIA-like (weak) thermotolerance phenotypes. Reference controls included on the blots are: farnesylated and uncleaved Ydj1p (CASQ); farnesylated, cleaved, and carboxymethylated Ydj1p (CVIA); unmodified Ydj1p (SASQ). Unmodified Ydj1p (open triangle) migrates at a larger apparent kDa than prenylated Ydj1p (closed triangle). The values in B reflect the thermotolerance scores observed for the corresponding mutant. (C) Yeast expressing the indicated a-factor Cxxx variants in a mfa1∆ mfa2∆ background were assessed for mating competence using a serial dilution mating assay (panel) and quantitative mating assay (values). Values represent mating efficiency relative to wild-type CVIA, which was set to 100%; values were determined using four or more replicates from two or more individual experiments. The panel is representative of one of the replicates.

Figure 4

Investigations of the geranylgeranylation potential of Ydj1p. (A) The indicated Ydj1p Cxxx variants were assessed for their ability to restore thermotolerance to a ydj1∆ ram1∆ (yWS2542) background as described for Figure 1, except that the 40° incubation was ∼80 hr without recovery. (B) Lysates for gel-shift assays were prepared from mid-log cultures incubated at either 40° or 25° (the latter are marked with an asterisk). The strain backgrounds used were ydj1∆ RAM1 (+; yWS2544) and ydj1∆ ram1∆ (−; yWS2542). Of note, the ram1∆ cultures were slow growing relative to RAM1 cultures at 40°, taking 36 or more hours instead of 18–24 hr to achieve the same cell density. Unmodified Ydj1p (open triangle) migrates at a larger apparent kDa than prenylated Ydj1p (closed triangle).

Figure 5

Frequency analysis of sequences identified by thermotolerance screening. Sequences were categorized by their associated thermotolerance scores (see Figure S2). For each grouping, a WebLogo analysis was performed (Crooks et al. 2004). Groupings were (A) all identified sequences (n = 153), (B) those most like CASQ (i.e., CASQ-like; T-score range 4.5–5; n = 82), (C) those most like CVIA (i.e., CVIA-like; T-score range 2.50–3.49; n = 14), (D) the subset of sequences from B that had a branched chain amino acid at x₂ (n = 10), and (E) sequences identified by a Ras-based strategy as having high likelihood of prenylation (enrichment score >3; n = 369) (Stein et al. 2015).

Figure 6

Amino acid frequency in hits recovered in Ydj1p- and Ras-based screens. The frequency occurrences of each amino acid at the x₁, x₂ and x₃ positions were calculated for the set of (A) Ydj1p and (B) Ras-based sequences. The complete set of Ydj1p-based sequences (n = 153) and the reduced-size set of Ras-based sequences (n = 369) were used for the analysis. Frequency values for Ydj1p-based sequences were normalized for codon bias (e.g., Leu codons are over-represented 6× relative to the Met codon); Ras-based sequences did not need normalization due to study design. Amino acids are clustered as reported for WebLogo analyses. Note that y-axis scales differ for the three panels in B.