Metaxin 1 interacts with metaxin 2, a novel related protein associated with the mammalian mitochondrial outer membrane

A recently described protein, metaxin 1, serves as a component of a preprot ein 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 p rotein, which we have termed metaxin 2, as a metaxin 1-binding protein. Met axin 2 shares 29% identity with metaxin 1 at the amino acid level, but meta xin 2, unlike metaxin 1, lacks a C-terminal mitochondrial outer membrane si gnal-anchor domain. Two C. elegans hypothetical proteins, CelZC97.1 and Cel F39B2.i, share high sequence similarity with metaxin 2 and metaxin 1, respe ctively, and likely represent the C. elegans orthologs. Affinity-purified a ntibodies against metaxin 2 were prepared against the recombinant protein p roduced 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 meta xin 2 cDNA, was found solely in mitochondria. Alkali extraction of mitochon dria indicated that metaxin 2 is peripherally associated with mitochondrial membranes. Metaxin 2 in intact mitochondria was susceptible to digestion w ith protei nase K, indicating that metaxin 2 is located on the cytosolic fa ce of the mitochondrial outer membrane. Finally, baculoviruses encoding a H is(6)-tagged metaxin 2 and an untagged metaxin 1 lacking its C-terminal tra nsmembrane domain were produced and used separately or in combination to in fect Sf21 insect cells. Metaxin 1 bound to a Ni2+-chelate affinity column o nly in the presence of metaxin 2, indicating that metaxin 1 and metaxin 2 i nteract when overexpressed in insect cells. These results suggest that meta xin 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 comp lex may play a role in protein import into mammalian mitochondria. J. Cell. Biochem. 74:11-22, 1999. (C) 1999 Wiley-Liss, Inc.