Towards the Systematic Mapping and Engineering of the Protein Prenylation Machinery in Saccharomyces cerevisiae

Abstract

Protein prenylation is a widespread and highly conserved eukaryotic post-translational modification that endows proteins with the ability to reversibly attach to intracellular membranes. The dynamic interaction of prenylated proteins with intracellular membranes is essential for their signalling functions and is frequently deregulated in disease processes such as cancer. As a result, protein prenylation has been pharmacologically targeted by numerous drug discovery programs, albeit with limited success. To a large extent, this can be attributed to an insufficient understanding of the interplay of different protein prenyltransferases and the combinatorial diversity of the prenylatable sequence space. Here, we report a high-throughput, growth-based genetic selection assay in Saccharomyces cerevisiae based on the Ras Recruitment System which, for the first time, has allowed us to create a comprehensive map of prenylatable protein sequences in S. cerevisiae. We demonstrate that potential prenylatable space is sparsely (6.2%) occupied leaving room for creation of synthetic orthogonal prenylatable sequences. To experimentally demonstrate that, we used the developed platform to engineer mutant farnesyltransferases that efficiently prenylate substrate motives that are not recognised by endogenous protein prenyltransferases. These uncoupled mutants can now be used as starting points for the systematic engineering of the eukaryotic protein prenylation machinery.

Rce1: mechanism and inhibition

“…Other reporters such as Ras could also be utilized. Intriguingly, the reported specificity preferences of Sc Rce1 observed with the a -factor reporter are similar to those inferred from a Ras-based yeast genetic screen intended to interrogate farnesyl transferase specificity ( Stein et al 2015 ). The Ras-based screen evaluated the farnesylation potential of all 8,000 possible CaaX variations.…”

“… Weblogo amino acid frequency analysis of sequences identified by a Ras-based strategy ( Stein et al 2015 ). The sequences analyzed were part of a set of sequences ( n = 496) having an enrichment score greater than 3, which is suggestive of prenylation.…”

Protein Lipidation: Occurrence, Mechanisms, Biological Functions, and Enabling Technologies

“…Using a yeast-based screening system for FT, randomization of aaX residues in the CaaX sequence motif showed that the second “a” strongly prefers small hydrophobic residues, whereas the first a and X have relatively more relaxed specificities. 68 This study further expanded the list of prenylated substrates.…”

Analogs of farnesyl diphosphate alter CaaX substrate specificity and reactions rates of protein farnesyltransferase

“…Alternatively, attaching the CaaX sequence or entire C-terminal domain of interest to newly developed reporter constructs allows for in cellulo tracking of prenylation with native FPP. 34,35 …”