Analysis of the functional domain of the rat liver mitochondrial import receptor Tom20.
Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins. It consists of the N-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster. To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Deltatom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import. Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild-type Tom20, whereas a mutant lacking the linker segment did not. In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69. Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not.
Iwahashi
J
J
Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812, Japan.
Yamazaki
S
S
Komiya
T
T
Nomura
N
N
Nishikawa
S
S
Endo
T
T
Mihara
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D017931:DNA Primers; D005656:Fungal Proteins / Q000737:chemistry; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000648:ultrastructure; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D026901:Membrane Transport Proteins; D008930:Mitochondria, Liver / Q000737:chemistry* / Q000648:ultrastructure; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000737:chemistry; D016133:Polymerase Chain Reaction; D051381:Rats; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013367:Submitochondrial Particles / Q000737:chemistry / Q000648:ultrastructure
D017931:DNA Primers; D005656:Fungal Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins; C489350:TOM20 protein, S cerevisiae; C084008:TOMM20 protein, human; C489998:Tomm20 protein, rat