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Functional Mapping of N-terminal Residues in the Yeast Proteome Uncovers Novel Determinants for Mitochondrial Protein Import

Salomé Nashed, Houssam El Barbry, Médine Benchouaia, Angélie Dijoux-Maréchal, Thierry Delaveau, Nadia Ruiz-Gutierrez, Lucie Gaulier, Déborah Tribouillard-Tanvier, Guillaume Chevreux, Stéphane Le Crom, Benoit Palancade, Frédéric Devaux, Elodie Laine, Mathilde Garcia

Abstract

N-terminal ends of polypeptides are critical for the selective co-translational recruitment of N-terminal modification enzymes. However, it is unknown whether specific N-terminal signatures differentially regulate protein fate according to their cellular functions. In this work, we developed an in-silico approach to detect functional preferences in cellular N-terminomes, and identified in S. cerevisiae more than 200 Gene Ontology terms with specific N-terminal signatures. In particular, we discovered that Mitochondrial Targeting Sequences (MTS) show a strong and specific over-representation at position 2 of hydrophobic residues known to define potential substrates of the N-terminal acetyltransferase NatC. We validated mitochondrial precursors as co-translational targets of NatC by selective purification of translating ribosomes, and found that their N-terminal signature is conserved in Saccharomycotina yeasts. 

Introduction

As soon as they emerge from the ribosomal tunnel, the protein nascent chains recruit factors that will play key roles in their life cycle [15]. Such recruitment is dependent on the nature of the protein N-terminal amino acid residues. In particular, the specific recognition and co-translational action of several N-terminal modification enzymes is determined by the type of the amino acid residue directly following the initiator methionine, namely the residue at position 2.

Materials and method

S. cerevisiae strains, plasmids, and growth conditions

All Saccharomyces cerevisiae strains used in this study are described in Supplementary Methods (Table A in S1 Supplementary Methods). The plasmids used for yeast transformation are also described in the Supplementary Methods (Table B in S1 Supplementary Methods) as well as the oligonucleotides used for the diverse genetic constructs (Table C and D in S1 Supplementary Methods).

Results

Detection of functional biases in amino acid usage at position 2 in S. cerevisiae proteome

The amino acid usage at position 2 of S. cerevisiae proteins (Fig 1A) is clearly different from the average amino acid distribution in its proteome. Strikingly, we observed a serine at this position in nearly 25% of the yeast proteins. This overrepresentation is both highly significant (HGT score equal to 268 corresponding to a p-value after hypergeometric test equal to 10−268, see Methods for a definition of HGT score) and specific to position 2 (aspecificity score of 0%, see Methods). By contrast, several amino acids are underrepresented (HGT score <-3 and aspecificity score <5%), including the hydrophobic residues leucine, isoleucine, and tyrosine as well as charged or polar residues such as arginine, glutamate and glutamine. Such asymmetric distribution, with the over-representation of serine, is conserved in budding yeasts (S1A Fig), as indicated by proteome analysis of 17 yeasts spanning nearly 400 million years of evolution of the Saccharomycotina lineage [26,27].

Discussion

In this work, we developed an in-silico approach to identify functional groups of proteins with N-terminal amino acid usage biases. The resulting functional mapping of N-terminal residues reveals possible evolutionary constraints at position 2 of nascent chains and highlights potential early mechanisms of protein regulation or targeting. In particular, we discovered the potential critical role of the residue located at position 2 of mitochondrial N-terminal targeting sequences in S. cerevisiae. This prediction was experimentally validated using an original system based on a dominant-negative allele of HSP60, that we set up to test the efficiency of mitochondrial import after mutagenesis at position 2 of the MTS. 

Acknowledgments

We are grateful to Jean-Paul Di Rago, Geneviève Dujardin and Naima Belgareh for the fruitful discussions on mitochondria and suggestions of experimental systems. Our thoughts go to the late Agnes Delahodde who provided us with the heat-sensitive yeast strain that proved crucial for the validation of our in-silico prediction.

Citation: Nashed S, El Barbry H, Benchouaia M, Dijoux-Maréchal A, Delaveau T, Ruiz-Gutierrez N, et al. (2023) Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import. PLoS Genet 19(8): e1010848. https://doi.org/10.1371/journal.pgen.1010848

Editor: Aimee M. Dudley, Pacific Northwest Research Institute, UNITED STATES

Received: November 14, 2022; Accepted: June 29, 2023; Published: August 16, 2023

Copyright: © 2023 Nashed et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: Scripts used to perform analyses of amino acid usage bias are available at https://tinyurl.com/mr2r54x8. Mass spectrometry proteomic data were deposited on the ProteomeXchange Consortium via the PRIDE partner repository with dataset ID PXD034922 (http://www.ebi.ac.uk/pride/archive/projects/PXD034922). The microarray data and the related protocols are available at the ArrayExpress website with the dataset identifiers E-MTAB-11772 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-11772/). All other relevant data are within the manuscript and its Supporting Information files.

Funding: M.G. received funding from the Sorbonne University Emergence program, and from the ARC Foundation for Cancer Research (https://www.fondation-arc.org/recherche-cancer, PJA 20171206624). A.D.-M. received a salary from Sorbonne University as part of the Emergence program. L.G. received a Master 2 scholarship from the Systems Biology Network of the Institute of biology Paris-Seine. H.E.B. received a PhD grant from the doctoral school Complexité Du Vivant of Sorbonne University, and S.N.'s PHD was funded by the program for disabled students of the Centre National de la Recherche Scientifique. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

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