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.