Evaluating protein prenylation of human and viral CaaX sequences using a humanized yeast system

ABSTRACT Prenylated proteins are prevalent in eukaryotic biology (∼1-2% of proteins) and are associated with human disease, including cancer, premature aging and infections. Prenylated proteins with a C-terminal CaaX sequence are targeted by CaaX-type prenyltransferases and proteases. To aid investigations of these enzymes and their targets, we developed Saccharomyces cerevisiae strains that express these human enzymes instead of their yeast counterparts. These strains were developed in part to explore human prenyltransferase specificity because of findings that yeast FTase has expanded specificity for sequences deviating from the CaaX consensus (i.e. atypical sequence and length). The humanized yeast strains displayed robust prenyltransferase activity against CaaX sequences derived from human and pathogen proteins containing typical and atypical CaaX sequences. The system also recapitulated prenylation of heterologously expressed human proteins (i.e. HRas and DNAJA2). These results reveal that substrate specificity is conserved for yeast and human farnesyltransferases but is less conserved for type I geranylgeranyltransferases. These yeast systems can be easily adapted for investigating the prenylomes of other organisms and are valuable new tools for helping define the human prenylome, which includes physiologically important proteins for which the CaaX modification status is unknown.

at chromosomal loci (no brackets) or on plasmids (brackets).Select strains were also transformed with empty vectors (vect) so that all strains had the same selectable markers.
Qualitative (SD and SC-Lys panels) and quantitative (values) mating tests were performed in parallel.For the qualitative mating assay, a representative result shown.For the quantitative mating assay, the value for the wildtype strain was set to 100% (condition 7), errors are standard error of the mean (SEM), and the number of biological replicates evaluated is indicated.Strains used were yWS3276 (1), yWS3277 (2), yWS3278 (3), yWS3408 (4), yWS3280 (5), yWS3282 (6), and yWS3283 (7).A detailed description of the assay is reported in Fig. 2B.B) Representative colony counts and calculations from a single quantitative mating test that contributed to the "% mating" data in panel A. See Materials and Methods and for additional assay details.C) HsFTase subunits were encoded behind the orthologous yeast gene promoters (i.e., PRAM2 and PRAM1) or the constitutive phosphoglycerate kinase 1 promoter (PPGK1) and introduced as URA3 and LEU2 marked plasmids into yWS3202 (MATa ram1∆).The qualitative mating assay was performed as in Panel A and Fig. 2B.The experiment was performed twice.
See Table S4 for plasmid details.D) Gel-shift analysis of Ydj1 using the strains described in panel A. Total cell lysates were prepared and analyzed as described in Fig. 2D.uunprenylated; pprenylated.The mating assay was performed as described in Fig. 2B using plasmid-encoded a-factor-CaaX variants transformed into a yeast strain lacking chromosomal copies of the a-factor genes (SM2331; MATa mfa1∆ mfa2∆).The strain expresses both yeast CaaX proteases Rce1 and Ste24, which have very similar specificities to their human counterparts (Mokry et al., 2009;Plummer et al., 2006).The viral source of the CaaX sequence is indicated above each specific sequence when applicable.Other CaaX sequences serve as controls: unprenylated (SVIA), prenylated and cleaved (CVIA), and prenylated but not cleaved (CASQ, CAHQ).Data are representative of 3 biological replicates.2, 3, 6 and 7).Growth on 5FOA at 37 °C depends on functional FTase, which requires α and FTase β subunits of the same species (6 and 7).

Detailed plasmid construction strategies
Various oligonucleotides were used in plasmid constructions (Table S5).Plasmids encoding a-factor-CaaX variants were created by co-transforming yeast with appropriate PCR-derived DNA fragments and pWS610 (MluI digest) followed by SC-uracil selection.Plasmids encoding Ydj1-CaaX variants were created similarly using pWS1132 (NheI and AflII digest) followed by SC-uracil selection.pWS965 was generated by ligation of a KpnI-SacI fragment encoding HsRCE1 from pWS335 into the same sites of pRS416.pWS1277 was derived by subcloning a PCR-derived fragment encoding the RAM2 gene, including 439 bp 5´ UTR and 637 bp 3´ UTR, into pRS416.pWS1278 was derived identically to pWS1277 but contained 1333 bp 3´ UTR.pWS1424 was generated by co-transforming yeast with a PCRderived DNA fragment encoding the HIS6-HsDNAJA2 cDNA sequence and pWS1132 (BsaBI and Nhe1 digest) followed by SC-uracil selection.The HIS6-HsDNAJA2 cDNA sequence was PCR amplified from plasmid #19546 with oligos oWS1005 and oWS1006.pWS1651 was derived by subcloning a PCR-derived fragment amplified from BY4241 encoding the CDC43 gene, including 415 bp 5´ UTR and 500 bp 3´ UTR, into pRS316.pWS1658 was derived by using the BamHI-XhoI fragment of pWS1655 and recombination-based methods for direct gene replacement of RAM2 in pWS1278 (NheI digest).pWS1660 was derived by using the BamHI-XhoI fragment of pWS1657 and recombination-based methods for direct gene replacement of CDC43 in pWS1651 (HindIII digest).pWS1659 was derived by using the BamHI-XhoI fragment of pWS1656 and recombination-based methods for direct gene replacement of RAM1 in pWS1767 (MscI digest).pWS1767 was derived by subcloning a PCR-derived fragment amplified from BY4241 encoding the RAM1 ORF, 491 bp 5´ UTR, and 275 bp 3´ UTR into pRS316.QuickChange was used to derive pWS1997-pWS2000 from pWS1961 and pWS2256 from pWS1424.Each of the yeast plasmids encoding human prenyltransferase subunits was also engineered for expression driven by the PGK1 promoter.Using recombination-based methods, the PCR amplified PGK1 promoter (590 bp) derived from pWS948 was used to replace the orthologous yeast promoters.pWS1861 was derived using pWS1658 (NotI-digest), pWS1862 using pWS1658 (AfeI-digest), pWS1863 using pWS1719 (PstI-digest), and pWS1914 using pWS1660 (BamHI-digest).pWS1861 and pWS1862 behave identically in all functional tests but differ in that pWS1862 retains 243 bp of RAM2 outlying genomic sequence upstream of the PGK1 promoter.

Fig. S1 .
Fig. S1.Interspecies complementation studies of FTase subunits: HsFTase and ScFTase subunits are not interchangeable, but co-expression of both α and β subunits of HsFTase can restore FTase activity in the absence of ScFTase.A) MATa haploid strains were engineered to express yeast (Sc) and/or human FTase α and β subunits (PGK1Hs), where subunits were encoded
Fig. S3.Gel-shift analysis of HSP40-CaaX variants with unusual gel mobilities.A) DNAJA2 exhibits major and minor bands by immunoblot.Plasmid-encoded DNAJA2 was produced in strains expressing ScFTase (Sc; yWS2544), HsFTase (Hs; yWS3186) or no FTase (∆; yWS3209).Total cell lysates were prepared and analyzed as described in Fig. 4E.The dashed line is aligned with the main band of unprenylated DNAJA2 to better visualize that the faint band above the main band of prenylated DNAJA2 does not comigrate with unprenylated DNAJA2.B) Ydj1-CKRC has aberrant gel mobility and is not farnesylated by HsFTase.Plasmid-encoded Ydj1-CaaX variants were produced in strains expressing HsFTase (Hs; yWS3186) or no FTase (∆; yWS3209).Total cell lysates were prepared and analyzed as described in Fig.2D.See TableS4for plasmid details.

Fig. S7 .
Fig. S7.Cartoon summaries of humanized prenyltransferase strains described in this study.The indicated strains are in addition to these reported in Figs.2A and 7A.The strain numbers, genomic architecture, associated plasmids, and relevant phenotypes are indicated on each cartoon.

Supplementary information Fig. S6. Active site architectures resulting from three-dimensional alignments of human, rat, and yeast prenyltransferase subunits.
Subunit residues in the yeast subunit that are positionally conserved but not sequence conserved in the equivalent human subunit are bold. a

Table S1 . Quantification of percent a prenylation by human and yeast prenyltransferases based on gel-shift analysis of indicated sequences in the context of the Ydj1 reporter.
::KAN Disease Models & Mechanisms: doi:10.1242/dmm.050516:Supplementary information Disease Models & Mechanisms • Supplementary information

Table S2 .
Human and viral proteins with Cxxx sequences investigated in this study.