Tma108, a putative M1 aminopeptidase, is a specific nascent chain-associated protein inSaccharomyces cerevisiae

Abstract

The discovery of novel specific ribosome-associated factors challenges the assumption that translation relies on standardized molecular machinery. In this work, we demonstrate that Tma108, an uncharacterized translation machinery-associated factor in yeast, defines a subpopulation of cellular ribosomes specifically involved in the translation of less than 200 mRNAs encoding proteins with ATP or Zinc binding domains. Using ribonucleoparticle dissociation experiments we established that Tma108 directly interacts with the nascent protein chain. Additionally, we have shown that translation of the first 35 amino acids of Asn1, one of the Tma108 targets, is necessary and sufficient to recruit Tma108, suggesting that it is loaded early during translation. Comparative genomic analyses, molecular modeling and directed mutagenesis point to Tma108 as an original M1 metallopeptidase, which uses its putative catalytic peptide-binding pocket to bind the N-terminus of its targets. The involvement of Tma108 in co-translational regulation is attested by a drastic change in the subcellular localization of ATP2 mRNA upon Tma108 inactivation. Tma108 is a unique example of a nascent chain-associated factor with high selectivity and its study illustrates the existence of other specific translation-associated factors besides RNA binding proteins.

Introns Protect Eukaryotic Genomes from Transcription-Associated Genetic Instability

“…Total RNAs were directly extracted from yeast cells disrupted by bead beating. For nascent mRNA analysis, chromatin pellets were isolated using a described procedure (Carrillo Oesterreich et al, 2010) and checked for their protein content by western blot analysis using the following antibodies: anti-Rpl1 (Delaveau et al, 2016), anti-Rpl3 (DSHB) and anti-Rpb1 (BioLegend). Total or chromatinassociated mRNAs were then purified using the Nucleospin RNA II kit (Macherey-Nagel) and reverse-transcribed with Superscript-II reverse transcriptase (Invitrogen).…”

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

“…For analysis of canonical ribosomes and NatA, NatB and NatC-associated ribosomes, ribonucleoparticle purifications from cells expressing proteinA-tagged baits (respectively Rpl16a, Ard1, Nat3 and Mak3) were performed essentially as described in (32). Briefly, frozen grindates obtained from cells grown in rich medium were homogenized in nine volumes of extraction buffer (20 mM Hepes pH 7.5, 110 mM KOAc, 2 mM MgCl 2 , 0.1% Tween-20, 0,5% Triton X−100, 1 mM DTT, 1× protease inhibitors cocktail, complete EDTA-free, Roche and antifoam B, Sigma, 1:5000).…”

“…3a, see also microarray data in Table S3). This approach had already proven sufficient sensitivity to study the selectivity of various nascent chain associated factors (36)(37)(38)(39). To validate our strategy, we also conducted sel-TRAP experiments on NatA and NatB, which preferences were previously confirmed by the impact of their deletion on N-terminal acetylome.…”

“…Inserted sequence Experiment pAEF-HSP60 (This Study *2 ) Cas9 sequence HSP60 Guide Sequence CRISPR/CAS9 mutagenesis of HSP60 genomic locus pZymA7 (32) ZWF1 promotor ASN1(1-1717)-13myc PGK1 terminator…”

Comparative Transcriptomics Highlights New Features of the Iron Starvation Response in the Human Pathogen Candida glabrata

“…The membrane was stripped by boiling 30 min in 62.5 mM Tris-HCl pH 6.8, SDS 2% and 4 mM DTT, followed by 10 washes in PBS-Tween (0.1%). Then, immunoblotting of the ribosomal protein Rpl3, used as a loading and transfer control, was performed using 1:10000 rabbit IgG Anti-Rpl3 [gift from M. Garcia: refer to ( Delaveau et al, 2016 )] and 1:15000 anti-rabbit IgG-HRP (Promega) as primary and secondary antibodies, respectively. Detection of the signals was performed using G:BOX Chemi XT4 (Syngene) following incubation with UptiLightTM HRP blot chemiluminescent ECL substrate (Interchim).…”