An Unusual Peptide Deformylase Features in the Human Mitochondrial N-terminal Methionine Excision Pathway

Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated. Dedicated machinery for N-terminal methionine excision (NME) was recently identified in plant organelles and shown to be essential in plastids. We report here the existence of mitochondrial NME in mammals, as shown by the identification of cDNAs encoding specific peptide deformylases (PDFs) and new methionine aminopeptidases (MAP1D). We cloned the two full-length human cDNAs and showed that the N-terminal domains of the encoded enzymes were specifically involved in targeting to mitochondria. In contrast to mitochondrial MAP1D, the human PDF sequence differed from that of known PDFs in several key features. We characterized the human PDF fully in vivo and in vitro. Comparison of the processed human enzyme with the plant mitochondrial PDF1A, to which it is phylogenetically related, showed that the human enzyme had an extra N-terminal domain involved in both mitochondrial targeting and enzyme stability. Mammalian PDFs also display non-random substitutions in the conserved motifs important for activity. Human PDF site-directed mutagenesis variants were studied and compared with the corresponding plant PDF1A variants. We found that amino acid substitutions in human PDF specifically altered its catalytic site, resulting in an enzyme intermediate between bacterial PDF1Bs and plant PDF1As. Because (i) human PDF was found to be active both in vitro and in vivo, (ii) the entire machinery is conserved and expressed in most animals, (iii) the mitochondrial genome expresses substrates for these enzymes, and (iv) mRNA synthesis is regulated, we conclude that animal mitochondria have a functional NME machinery that can be regulated. The N-terminal methionine excision (NME) 1The abbreviations used are: NMEN-terminal methionine excisionCDcatalytic domainDAPI4′-6-diamidino-2-phenylindoleFoN-formylGFPgreen fluorescent proteinHEKhuman embryonic kidneyEcE. coliHsH. sapiensMAPmethionine aminopeptidaseORFopen reading framePDFpeptide deformylasePDFIpeptide deformylase inhibitorspNAp-nitroanilideRFPred fluorescent proteinTCEPTris(2-carboxyethyl)-phosphineAtA. thaliana.1The abbreviations used are: NMEN-terminal methionine excisionCDcatalytic domainDAPI4′-6-diamidino-2-phenylindoleFoN-formylGFPgreen fluorescent proteinHEKhuman embryonic kidneyEcE. coliHsH. sapiensMAPmethionine aminopeptidaseORFopen reading framePDFpeptide deformylasePDFIpeptide deformylase inhibitorspNAp-nitroanilideRFPred fluorescent proteinTCEPTris(2-carboxyethyl)-phosphineAtA. thaliana. pathway is an essential pathway that removes the initial Met from two-thirds of the proteins of any proteome (1Meinnel T. Mechulam Y. Blanquet S. Biochimie (Paris). 1993; 75: 1061-1075Crossref PubMed Scopus (211) Google Scholar). Methionine aminopeptidase (MAP; EC 3.4.11.18) has been shown to be involved in this process in all organisms studied, and this activity facilitates subsequent protein modification in Eukaryotes (2Polevoda B. Sherman F. J. Mol. Biol. 2003; 325: 595-622Crossref PubMed Scopus (348) Google Scholar, 3Boisson B. Giglione C. Meinnel T. J. Biol. Chem. 2003; 278: 43418-43429Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). In Eubacteria, plastids, and mitochondria, the N-terminal Met residue of nascent polypeptides carries an N-formyl (Fo) group whereas in Eukaryotes and Archaea, nascent proteins synthesized in the cytoplasm start with a free Met (4Kozak M. Microbiol. Reviews. 1983; 47: 1-45Crossref PubMed Google Scholar). The N-terminal Met must be exposed to allow MAP activity in Eubacteria, and this is achieved by systematic removal of the Fo group by peptide deformylase (PDF; EC 3.5.1.88). PDF activity was long considered to be unique to this kingdom (5Mazel D. Pochet S. Marliere P. J. PubMed Scopus Google Scholar). have that PDF in most animals, and organisms C. M. Meinnel T. Mol. Microbiol. PubMed Scopus Google Scholar, C. M. B. Meinnel T. J. PubMed Scopus Google Scholar, T. Full Text Full Text PDF PubMed Scopus Google Scholar). have been shown to display peptide deformylase activity in both in vitro and in the have an N-terminal The N-terminal domain the proteins to the and mitochondria in C. M. B. Meinnel T. J. PubMed Scopus Google Scholar, C. Meinnel T. Full Text Full Text PDF PubMed Scopus Google Scholar, C. Meinnel T. J. 2003; PubMed Scopus Google Scholar, Giglione C. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar). it has been that the PDF of to the an essential T. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. D. D. PubMed Scopus Google Scholar, C. Meinnel T. PubMed Scopus Google Scholar). a that specifically both bacterial and plant PDFs C. Meinnel T. J. 2003; PubMed Scopus Google Scholar, C. J. PubMed Scopus Google Scholar). in vivo with this have shown that PDF is essential in and Giglione C. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar, C. J. PubMed Scopus Google Scholar, J. S. Full Text Full Text PDF PubMed Google Scholar). In plastids, NME a in the of a protein C. Meinnel T. J. 2003; PubMed Scopus Google Scholar). The was of Met was N-terminal methionine excision catalytic domain N-formyl fluorescent protein human embryonic methionine aminopeptidase reading peptide deformylase peptide deformylase fluorescent protein thaliana. N-terminal methionine excision catalytic domain N-formyl fluorescent protein human embryonic methionine aminopeptidase reading peptide deformylase peptide deformylase fluorescent protein thaliana. of a of Chem. PubMed Scopus Google Scholar). of have been to Full Text Full Text PDF PubMed Scopus Google Scholar). in and in the cytoplasm of whereas have been found in Eubacteria, in the cytoplasm of Eukaryotes and in the organelles of and C. M. B. Meinnel T. J. PubMed Scopus Google Scholar). The of and have these two enzymes display active have a N-terminal that is found in bacterial the catalytic domain to the enzymes also have an a conserved involved in catalytic this domain is from the and is essential for with J. PubMed Scopus Google Scholar). PDFs a of enzymes to the of C. M. Meinnel T. Mol. Microbiol. PubMed Scopus Google Scholar). PDFs of amino that the active T. C. S. Blanquet S. J. Mol. Biol. PubMed Scopus Google Scholar). the In most an is by of motifs and and a in activity D. J. Chem. Google Scholar, D. S. PubMed Scopus Google Scholar). is and several and have been that deformylase activity in C. M. Meinnel T. Mol. Microbiol. PubMed Scopus Google Scholar). The be by resulting in a active PDFs have to catalytic T. Blanquet S. J. PubMed Google Scholar, S. Blanquet S. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar). of PDF have been identified the of and sequence C. M. Meinnel T. Mol. Microbiol. PubMed Scopus Google Scholar, C. Meinnel T. PubMed Scopus Google Scholar, M. Giglione C. M. P. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar). The in several of active conserved and and PDF enzymes found in In contrast to PDF have deformylase to amino acid substitutions in motifs to C. Meinnel T. PubMed Scopus Google Scholar, P. C. S. M. J. PubMed Scopus Google Scholar). of the specific amino of the motifs to be for enzyme activity and as by site-directed mutagenesis by in vivo and in vitro T. C. S. Blanquet S. J. Mol. Biol. PubMed Scopus Google Scholar, T. C. Blanquet S. J. Mol. Biol. PubMed Scopus Google Scholar, T. C. F. Blanquet S. PubMed Scopus Google Scholar, S. P. C. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar, F. S. C. Blanquet S. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar). two of for PDF enzymes found in and PDFs to both and mitochondria C. M. B. Meinnel T. J. PubMed Scopus Google Scholar, C. Meinnel T. Full Text Full Text PDF PubMed Scopus Google Scholar). The and of the PDFs of and Giglione C. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar, M. Giglione C. M. P. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar, S. B. S. D. Full Text Full Text PDF PubMed Scopus Google Scholar). PDFs plant mitochondrial PDFs and PDF from C. Meinnel T. Full Text Full Text PDF PubMed Scopus Google Scholar). of plant has shown that from most studied PDF1Bs and in of the Giglione C. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar). In this we that the specific and functional PDF and MAP1D, is expressed and to animal mitochondria. mutagenesis with and showed that from characterized PDFs in of its be used as the for for PDF for in the of bacterial in and enzymes for protein were from The used have been S. P. C. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar, T. S. Blanquet S. PubMed Scopus Google Scholar). for were from were synthesized by was with and enzyme we used and all were with in S. Blanquet S. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar). the of all PDF were to the as M. Giglione C. M. P. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar). of residue in that the the residue corresponding to amino acid in as shown in the in from 1The abbreviations used are: NMEN-terminal methionine excisionCDcatalytic domainDAPI4′-6-diamidino-2-phenylindoleFoN-formylGFPgreen fluorescent proteinHEKhuman embryonic kidneyEcE. coliHsH. sapiensMAPmethionine aminopeptidaseORFopen reading framePDFpeptide deformylasePDFIpeptide deformylase inhibitorspNAp-nitroanilideRFPred fluorescent proteinTCEPTris(2-carboxyethyl)-phosphineAtA. thaliana. of to and a N-terminal methionine excision catalytic domain N-formyl fluorescent protein human embryonic methionine aminopeptidase reading peptide deformylase peptide deformylase fluorescent protein thaliana. and have been T. Blanquet S. J. PubMed Google Scholar, M. M. Microbiol. PubMed Google Scholar). was from and a for and The human was from was in with were by site-directed the with the site-directed mutagenesis were by the with a in and and with were between the and of The F. P. M. Mol. Biol. PubMed Scopus Google the N-terminal mitochondrial domain of to which was used as a for mitochondrial We the of the between the unique as C. M. B. Meinnel T. J. PubMed Scopus Google Scholar). of the was a encoding of between and of with an N-terminal and a the of the catalytic domain of of We a the for subsequent the N-terminal targeting sequence of synthesized in were by in vitro as T. Mechulam Y. PubMed Scopus Google Scholar). The initial involved of the of with to We between the and of to the full-length The sequence of encoding full-length is from a to the to of the a of the PDF sequence the the synthesis of this protein D. J. J. PubMed Scopus Google Scholar). The which is to several C. C. J. B. 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D. 2003; PubMed Scopus Google Scholar). of the of the human mitochondrial genome to be substrates of to the known of MAP in T. Mechulam Y. Blanquet S. Biochimie (Paris). 1993; 75: 1061-1075Crossref PubMed Scopus (211) Google Scholar). The of these and and and The for conserved in all encoded in organisms in which a has been animal PDFs and enzyme found in mitochondrial to have from PubMed Scopus Google Scholar). both mitochondrial NME from an from an to an of be to that the of as the were 2003; PubMed Scopus Google is to mitochondrial to the identification of a mitochondrial MAP the of and Full Text PDF PubMed Scopus Google Scholar). to the of the were from by and to a and NME in Human NME has been shown to in vivo in by (i) the of machinery the initial Met C. M. B. Meinnel T. J. PubMed Scopus Google and (ii) the of a of N-terminal protein in C. Meinnel T. Full Text Full Text PDF PubMed Scopus Google Scholar). phylogenetically to plant and C. M. B. Meinnel T. J. 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The residue to and is residue a in and an in human We the of this by a of the active of that a be in between the of and The in that residue is a residue is residue is as in residue is We conclude that the substitutions of and in animal PDFs and that these substitutions it to PDF activity in mitochondria, to to the in this we the that these with the N-terminal of of the of Human PDF for PDF PDF of the most of new C. M. Meinnel T. Mol. Microbiol. PubMed Scopus Google Scholar, J. 2003; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). of a functional PDF in a as to the of in human the mitochondrial of this to and for the of to a of also to plant Giglione C. Meinnel T. J. Mol. Biol. PubMed Scopus Google Scholar). to most and as an for this a of that mitochondrial used in human as was the of in the long to of We that be to the of these in this that animal PDFs intermediate between and PDF1Bs in of animal PDFs to that whereas plant Giglione C. Meinnel T. J. Mol. Biol. 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