Functional mapping of N-terminal residues in the yeast proteome uncovers novel determinants for mitochondrial protein import

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. Finally, systematic mutagenesis of the position 2 in a prototypal yeast mitochondrial protein confirmed its critical role in mitochondrial protein import. Our work highlights the hydrophobicity of MTS N-terminal residues and their targeting by NatC as important features for the definition of the mitochondrial proteome, providing a molecular explanation for mitochondrial defects observed in yeast or human NatC-depleted cells. Functional mapping of N-terminal residues thus has the potential to support the discovery of novel mechanisms of protein regulation or targeting.

*4 X represents the amino acid that replaces leucine at position 2 of Hsp60p.This modification was first introduced into the S. cerevisiae genome with CRISPR/CAS9 technology using HSP60 repair cassettes that include the desired mutations (see Table S3 and S4).The mutated HSP60 sequences, including its promoter (680 bp upstream of the initiator codon), were then amplified by PCR (see oligonucleotides used in Table S3) and inserted between the SacI and PacI restriction sites into the plasmid pZMYA7 in fusion with the 13myc sequence.The forward and reverse HSP60 guide oligonucleotides were hybridized and the resulting dsDNA was inserted in LguI site (LguI extensions underlined in sequences) of the pAEF5 plasmid (2).The resulting plasmid (pAEF HSP60) was co-transformed in yeast (YPH499 or YPH499 pam16Δ-MAGN76D strain) with the repair cassette previously obtained by hybridization of the forward and reverse oligonucleotides K7HSP60.In these oligonucleotides, the codon following the initiator methionine (underlined in the sequence) was replaced with codons encoding the desired X mutation (indicated by YYY/ZZZ in the sequence, see Table S4 for the codon chosen for each mutation).In the repair cassette, a synonymous mutation was introduced into the HSP60 sequence to eliminate the Pam site targeted by the CAS9 endonuclease with the chosen guide RNA sequence.

Table D: Codon used in the reparation cassette to encode the desired X mutation
The selected codons are not rare codons in S. cerevisiae.Phosphorylation (STY).Spectra were filtered using a 1% FDR with the percolator node.

X
formic acid) followed by column regeneration, giving a total time of 120 minutes.Precursor peptides were analyzed in the Orbitrap cell in positive mode, at a resolution of 70,000, with a mass range of m/z 375-1500 and an AGC target of 3.10 6 .MS/MS data were acquired in the Orbitrap cell in a Top20 datadependent mode with a dynamic exclusion of 30 seconds.Fragments were obtained by Higher-energy C-trap Dissociation (HCD) activation with a collisional energy of 27% and a quadrupole isolation window of 1.4 Da.The Orbitrap cell was set at a resolution of 17,500, m/z 200-2000 and an AGC target of 2.10 5 .Peptides with unassigned charge states or monocharged were excluded from the MS/MS acquisition.The maximum ion accumulation times were set to 50 ms for MS acquisition and 45 ms for MS/MS acquisition.Data were processed with Proteome Discoverer 2.2 software (Thermo Fisher scientific, San Jose, CA) coupled to an in-house Mascot search server (Matrix Science, Boston, MA; version 2.5.1).MS/MS spectra were searched against the SwissProt protein database release 2017_09 with the Saccharomyces Cerevisiae (baker's yeast) taxonomy and a maximum of 2 missed cleavages.Precursor and fragment mass tolerances were set to 6 ppm and 0.02 Da respectively.The following posttranslational modifications were included as variable: Acetyl (Protein N-term), Oxidation (M),

Table B : Plasmids and derived strains
1Strains tagged at their C terminus were obtained from BY4741 strain, after classical procedure of lithium acetate transformation, by homologous recombination with ProtA-His5 cassette amplified from pBXA (Rout et al., 2000 , provided by M. Rout, The Rockefeller, University, New York, NY) * 2 Mak3 deletions were obtained by homologous recombination with the KanMX4 deletion cassette amplified from the BY4741 mak3Δ strain

Table E : List of the yeast species used for the genomic comparative studies
Digestion was performed overnight at 37°C in the presence of 12.5 µg/ml of sequencing grade trypsin (Promega, Madison, Wi, USA).Peptides mixtures were analyzed by a Q-Exactive Plus coupled to a Nano-LC Proxeon 1000 (both from Thermo Scientific).Peptides were separated by chromatography