Dataset Information

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.

SUBMITTER: Berger BM

PROVIDER: S-EPMC6283164 | biostudies-other | 2018 Dec

REPOSITORIES: biostudies-other

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

Berger Brittany M BM Kim June H JH Hildebrandt Emily R ER Davis Ian C IC Morgan Michael C MC Hougland James L JL Schmidt Walter K WK

Genetics 20180926 4

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 in ...[more]

PMID: 30257935

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Project description:Maturation of the Saccharomyces cerevisiae a-factor precursor involves COOH-terminal CAAX processing (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) followed by cleavage of an NH2-terminal extension (two sequential proteolytic processing steps). The aim of this study is to clarify the precise role of Ste24p, a membrane-spanning zinc metalloprotease, in the proteolytic processing of the a-factor precursor. We demonstrated previously that Ste24p is necessary for the first NH2-terminal processing step by analysis of radiolabeled a-factor intermediates in vivo (Fujimura-Kamada, K., F.J. Nouvet, and S. Michaelis. 1997. J. Cell Biol. 136:271-285). In contrast, using an in vitro protease assay, others showed that Ste24p (Afc1p) and another gene product, Rce1p, share partial overlapping function as COOH-terminal CAAX proteases (Boyartchuk, V.L., M.N. Ashby, and J. Rine. 1997. Science. 275:1796-1800). Here we resolve these apparently conflicting results and provide compelling in vivo evidence that Ste24p indeed functions at two steps of a-factor maturation using two methods. First, direct analysis of a-factor biosynthetic intermediates in the double mutant (ste24Delta rce1Delta) reveals a previously undetected species (P0*) that fails to be COOH terminally processed, consistent with redundant roles for Ste24p and Rce1p in COOH-terminal CAAX processing. Whereas a-factor maturation appears relatively normal in the rce1Delta single mutant, the ste24Delta single mutant accumulates an intermediate that is correctly COOH terminally processed but is defective in cleavage of the NH2-terminal extension, demonstrating that Ste24p is also involved in NH2-terminal processing. Together, these data indicate dual roles for Ste24p and a single role for Rce1p in a-factor processing. Second, by using a novel set of ubiquitin-a-factor fusions to separate the NH2- and COOH-terminal processing events of a-factor maturation, we provide independent evidence for the dual roles of Ste24p. We also report here the isolation of the human (Hs) Ste24p homologue, representing the first human CAAX protease to be cloned. We show that Hs Ste24p complements the mating defect of the yeast double mutant (ste24Delta rce1Delta) strain, implying that like yeast Ste24p, Hs Ste24p can mediate multiple types of proteolytic events.

Project description:We present here the 2011 update of the AutoMotif Service (AMS 4.0) that predicts the wide selection of 88 different types of the single amino acid post-translational modifications (PTM) in protein sequences. The selection of experimentally confirmed modifications is acquired from the latest UniProt and Phospho.ELM databases for training. The sequence vicinity of each modified residue is represented using amino acids physico-chemical features encoded using high quality indices (HQI) obtaining by automatic clustering of known indices extracted from AAindex database. For each type of the numerical representation, the method builds the ensemble of Multi-Layer Perceptron (MLP) pattern classifiers, each optimising different objectives during the training (for example the recall, precision or area under the ROC curve (AUC)). The consensus is built using brainstorming technology, which combines multi-objective instances of machine learning algorithm, and the data fusion of different training objects representations, in order to boost the overall prediction accuracy of conserved short sequence motifs. The performance of AMS 4.0 is compared with the accuracy of previous versions, which were constructed using single machine learning methods (artificial neural networks, support vector machine). Our software improves the average AUC score of the earlier version by close to 7 % as calculated on the test datasets of all 88 PTM types. Moreover, for the selected most-difficult sequence motifs types it is able to improve the prediction performance by almost 32 %, when compared with previously used single machine learning methods. Summarising, the brainstorming consensus meta-learning methodology on the average boosts the AUC score up to around 89 %, averaged over all 88 PTM types. Detailed results for single machine learning methods and the consensus methodology are also provided, together with the comparison to previously published methods and state-of-the-art software tools. The source code and precompiled binaries of brainstorming tool are available at http://code.google.com/p/automotifserver/ under Apache 2.0 licensing.

Dataset Information

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

Publications

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

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