title: Comparative Analysis of Mitochondrial N-Termini from Mouse, Human, and Yeast.
abstract: The majority of mitochondrial proteins are encoded in the nuclear genome, translated in the cytoplasm, and directed to the mitochondria by an N-terminal presequence that is cleaved upon import. Recently, N-proteome catalogs have been generated for mitochondria from yeast and from human U937 cells. Here, we applied the subtiligase method to determine N-termini for 327 proteins in mitochondria isolated from mouse liver and kidney. Comparative analysis between mitochondrial N-termini from mouse, human, and yeast proteins shows that whereas presequences are poorly conserved at the sequence level, other presequence properties are extremely conserved, including a length of ∼20-60 amino acids, a net charge between +3 to +6, and the presence of stabilizing amino acids at the N-terminus of mature proteins that follow the N-end rule from bacteria. As in yeast, ∼80% of mouse presequence cleavage sites match canonical motifs for three mitochondrial peptidases (MPP, Icp55, and Oct1), whereas the remainder do not match any known peptidase motifs. We show that mature mitochondrial proteins often exist with a spectrum of N-termini, consistent with a model of multiple cleavage events by MPP and Icp55. In addition to analysis of canonical targeting presequences, our N-terminal dataset allows the exploration of other cleavage events and provides support for polypeptide cleavage into two distinct enzymes (Hsd17b4), protein cleavages key for signaling (Oma1, Opa1, Htra2, Mavs, and Bcs2l13), and in several cases suggests novel protein isoforms (Scp2, Acadm, Adck3, Hsdl2, Dlst, and Ogdh). We present an integrated catalog of mammalian mitochondrial N-termini that can be used as a community resource to investigate individual proteins, to elucidate mechanisms of mammalian mitochondrial processing, and to allow researchers to engineer tags distally to the presequence cleavage.
authors.affiliation: From the ‡Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114; scalvo@broadinstitute.org.|§Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.|¶Broad Institute, Cambridge, Massachusetts 02141.
authors.forename: Sarah E
authors.identifier: 0000-0001-6449-6058
authors.initials: SE
authors.lastname: Calvo
authors.affiliation: **Departments of Pharmaceutical Chemistry and.
authors.forename: Olivier
authors.identifier: 0000-0001-7068-7299
authors.initials: O
authors.lastname: Julien
authors.affiliation: ¶Broad Institute, Cambridge, Massachusetts 02141.
authors.forename: Karl R
authors.initials: KR
authors.lastname: Clauser
authors.affiliation: From the ‡Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114.|§Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Hongying
authors.initials: H
authors.lastname: Shen
authors.affiliation: From the ‡Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114.|§Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Kimberli J
authors.initials: KJ
authors.lastname: Kamer
authors.affiliation: **Departments of Pharmaceutical Chemistry and.|§§Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143.
authors.forename: James A
authors.initials: JA
authors.lastname: Wells
authors.affiliation: From the ‡Howard Hughes Medical Institute, Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114.|§Department of Systems Biology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: Vamsi K
authors.identifier: 0000-0001-9924-642X
authors.initials: VK
authors.lastname: Mootha
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D019143:Evolution, Molecular; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D051379:Mice; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins