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Towards the systematic mapping and engineering of the protein prenylation machinery in Saccharomyces cerevisiae.

Stein, Viktor and Kubala, Marta H. and Steen, Jason and Grimmond, Sean M. and Alexandrov, Kirill (2015):
Towards the systematic mapping and engineering of the protein prenylation machinery in Saccharomyces cerevisiae.
In: PloS one, pp. e0120716, 10, (3), ISSN 1932-6203, [Article]

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.

Item Type: Article
Erschienen: 2015
Creators: Stein, Viktor and Kubala, Marta H. and Steen, Jason and Grimmond, Sean M. and Alexandrov, Kirill
Title: Towards the systematic mapping and engineering of the protein prenylation machinery in Saccharomyces cerevisiae.
Language: English
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.

Journal or Publication Title: PloS one
Volume: 10
Number: 3
Divisions: 10 Department of Biology
10 Department of Biology > Protein Engineering of Ion Conducting Nanopores
Date Deposited: 14 Nov 2016 11:30
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