title: Metaxin 1 interacts with metaxin 2, a novel related protein associated with the mammalian mitochondrial outer membrane.
abstract: A recently described protein, metaxin 1, serves as a component of a preprotein import complex in the outer membrane of the mammalian mitochondrion. A yeast two-hybrid screen with metaxin 1 as bait has now identified a novel protein, which we have termed metaxin 2, as a metaxin 1-binding protein. Metaxin 2 shares 29% identity with metaxin 1 at the amino acid level, but metaxin 2, unlike metaxin 1, lacks a C-terminal mitochondrial outer membrane signal-anchor domain. Two C. elegans hypothetical proteins, CelZC97.1 and CelF39B2.i, share high sequence similarity with metaxin 2 and metaxin 1, respectively, and likely represent the C. elegans orthologs. Affinity-purified antibodies against metaxin 2 were prepared against the recombinant protein produced in E. coli and were used to analyze the subcellular distribution of metaxin 2. In subcellular fractions of mouse liver, a 29 kD immunoreactive protein, consistent in size with the predicted translation product of metaxin 2 cDNA, was found solely in mitochondria. Alkali extraction of mitochondria indicated that metaxin 2 is peripherally associated with mitochondrial membranes. Metaxin 2 in intact mitochondria was susceptible to digestion with proteinase K, indicating that metaxin 2 is located on the cytosolic face of the mitochondrial outer membrane. Finally, baculoviruses encoding a His6-tagged metaxin 2 and an untagged metaxin 1 lacking its C-terminal transmembrane domain were produced and used separately or in combination to infect Sf21 insect cells. Metaxin 1 bound to a Ni2+-chelate affinity column only in the presence of metaxin 2, indicating that metaxin 1 and metaxin 2 interact when overexpressed in insect cells. These results suggest that metaxin 2 is bound to the cytosolic face of the mitochondrial outer membrane by means of its interaction with membrane-bound metaxin 1, and that this complex may play a role in protein import into mammalian mitochondria.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle 98195-7350, USA.
authors.forename: L C
authors.initials: LC
authors.lastname: Armstrong
authors.forename: A J
authors.initials: AJ
authors.lastname: Saenz
authors.forename: P
authors.initials: P
authors.lastname: Bornstein
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D007425:Intracellular Membranes / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D033681:Mitochondrial Membrane Transport Proteins; C490556:Mtx1 protein, mouse; D011506:Proteins; D011994:Recombinant Proteins