A comprehensivein vivoscreen of yeast farnesyltransferase activity reveals broad reactivity across a majority of CXXX sequences

Abstract

The basis for the current understanding of farnesyltransferase (FTase) specificity was pioneered through investigations of reporters like Ras and Ras-related proteins that possess a C-terminal CaaX motif that consists of 4 amino acid residues: Cysteine – aliphatic1– aliphatic2– variable (X). These studies led to the finding that proteins possessing the CaaX motif are subject to a 3-step post-translational modification pathway involving farnesylation, proteolysis, and carboxylmethylation. However, emerging evidence indicates that FTase can farnesylate sequences outside the canonical CaaX motif and that these motifs are only subject to farnesylation. This work investigates the scope of yeast FTase specificity through the use of genetic and next-generation sequencing methods using the reporter Ydj1, an HSP40 chaperone that only requires farnesylation for its activity. Our results support a wide array of sequence targets beyond the CaaX consensus and expand the potential target space of FTase within the yeast proteome.

A Humanized Yeast System for Evaluating the Protein Prenylation of a Wide Range of Human and Viral CaaX Sequences

“…Yeast expressing Ydj1 with prenylation-only sequences (CASQ and CAHQ) supported the most robust thermotolerance; these sequences were derived from yeast Ydj1 and human DNAJA2, respectively. The prenylation-only status of CAHQ was previously based on homology and prediction algorithms (Hildebrandt et al, 2016b; Kim et al, 2023) and is confirmed here by the observation that CAHQ supports thermotolerance similar to CASQ and is resistant to proteolysis by the CaaX proteases ( Supplemental Figure S3 ). Yeast expressing Ydj1 with canonical CaaX sequences (CVIA and CTLM) exhibited partial growth under this condition as has been previously observed (Hildebrandt et al, 2016b); these sequences were derived from yeast a -factor and Gγ Ste18, respectively.…”

“…The ability of Sc FTase to modify the non-canonical CaaX sequence associated with Ydj1 (CASQ) and other non-canonical sequences has been reported (Berger et al, 2018; Hildebrandt et al, 2016b; Kim et al, 2023; Ravishankar et al, 2023). The complementation studies described above indicate that Hs FTase can also modify the Ydj1 CASQ sequence (see Figure 1 ).…”

“…In yeast, the Ras2 GTPase and a -factor mating pheromone are highly studied examples of canonically modified CaaX proteins. In addition, CaaX sequences lacking a 1 and a 2 aliphatic residues can be farnesylated (Kim et al, 2023). These so-called shunted CaaX sequences retain their last three amino acids, resulting in a biochemically distinct C-terminus compared to canonically modified CaaX proteins (Hildebrandt et al, 2016b).…”

“…Methods for case-by-case verification of protein prenylation include radiolabeling with 3 H-mevalonate, gel mobility analysis, localization studies and mass spectrometry (Anderegg et al, 1988; Hancock et al, 1989; Hildebrandt et al, 2016b; Michaelson et al, 2005; Ravishankar et al, 2023). Methods for systematic verification have relied on metabolic labeling with reagents compatible with click chemistry, peptide arrays, and genetic approaches (Kho et al, 2004; Kim et al, 2023; Rashidian et al, 2013; Storck et al, 2019; Suazo et al, 2018; Suazo et al, 2021; Wang et al, 2014). Differentiating FTase and GGTase-I target specificity has mostly relied on in vitro and genetic approaches (Hougland et al, 2010; Kim et al, 2023; Stein et al, 2015).…”

“…Methods for systematic verification have relied on metabolic labeling with reagents compatible with click chemistry, peptide arrays, and genetic approaches (Kho et al, 2004; Kim et al, 2023; Rashidian et al, 2013; Storck et al, 2019; Suazo et al, 2018; Suazo et al, 2021; Wang et al, 2014). Differentiating FTase and GGTase-I target specificity has mostly relied on in vitro and genetic approaches (Hougland et al, 2010; Kim et al, 2023; Stein et al, 2015). Independent of the methods employed, it remains a distinct challenge to confirm the predictive prenylation of candidate CaaX sequences, especially across species where conservation of target specificity has not necessarily been confirmed.…”