Characterization of the novel mitochondrial protein import component, Tom34, in mammalian cells.
Tom34 is a newly-found component of the mitochondrial protein import machinery in mammalian cells with no apparent counterpart in fungi. RNA blot and immunoblot analyses showed that the expression of Tom34 varies among tissues and differs from that of the core translocase component Tom20. In contrast to a previous report [Nuttal, S.D. et al. (1997) DNA Cell Biol. 16, 1067-1074], the present study using a newly-prepared anti-Tom34 antibody with a high titer showed that Tom34 is present largely in the cytosolic fraction and partly in the mitochondrial and membrane fractions after fractionation of tissues and cells, and that the membrane-associated form is largely extractable with 0.1 M sodium carbonate. The in vitro import of preproteins into isolated rat mitochondria was strongly inhibited by DeltahTom34 which lacks the NH2-terminal hydrophobic region of human Tom34 (hTom34). Import was also strongly inhibited by anti-hTom34. In pulse-chase experiments using COS-7 cells, pre-ornithine transcarbamylase (pOTC) was rapidly processed to the mature form. Coexpression of hTom34 resulted in a stimulation of pOTC processing, whereas the coexpression of hTom34 antisense RNA caused inhibition. The results confirm that Tom34 plays a role in mitochondrial protein import in mammals, and suggest it to be an ancillary component of the translocation machinery in mammalian cells.
Chewawiwat
N
N
Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto, 860-0811, Japan.
Yano
M
M
Terada
K
K
Hoogenraad
N J
NJ
Mori
M
M
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004792:Enzyme Precursors / Q000378:metabolism; D005260:Female; D006367:HeLa Cells; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008566:Membranes / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D009954:Ornithine Carbamoyltransferase / Q000378:metabolism; D011247:Pregnancy; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013552:Swine; D014018:Tissue Distribution
D002352:Carrier Proteins; D017931:DNA Primers; D004792:Enzyme Precursors; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C084008:TOMM20 protein, human; C108485:TOMM34 protein, human; C489998:Tomm20 protein, rat; D009954:Ornithine Carbamoyltransferase