Metaxin, a novel gene located between the glucocerebrosidase and thrombospondin 
3 genes in the mouse, is essential for survival of the postimplantation mouse 
embryo. In this study, the subcellular location, domain structure, and 
biochemical function of metaxin were investigated. Anti-recombinant metaxin 
antibodies recognized 35- and 70-kDa proteins in mitochondria from various 
tissues; the 35-kDa protein is consistent in size with the predicted translation 
product of metaxin cDNA. When metaxin cDNA was transfected into COS cells, 
immunofluorescence staining demonstrated that the protein is located in 
mitochondria. Metaxin contains a putative mitochondrial outer membrane signal 
anchor domain at its C terminus, and a truncated form of metaxin lacking this 
signal anchor domain had a reduced association with mitochondria. In addition, 
metaxin was highly susceptible to proteases in intact mitochondria. We therefore 
conclude that metaxin is a mitochondrial protein that extends into the cytosol 
while anchored into the outer membrane at its C terminus. In its N-terminal 
region, metaxin shows significant sequence identity to Tom37, a component of the 
outer membrane portion of the mitochondrial preprotein translocation apparatus 
in Saccharomyces cerevisiae, but important structural differences, including 
apparently different mechanisms of targeting to membranes, also exist between 
the two proteins. Given the similar subcellular locations of metaxin and Tom37, 
the possible role of metaxin in mitochondrial preprotein import was 
investigated. Antibodies against metaxin, when preincubated with mitochondria, 
partially inhibited the uptake of radiolabeled preadrenodoxin into mitochondria. 
Metaxin is therefore the second mammalian component of the protein translocation 
apparatus of the mitochondrial outer membrane to be characterized at the 
molecular level and the first for which an inherited mutation has been 
described. The early embryonic lethal phenotype of mice lacking metaxin 
demonstrates that efficient import of proteins into mitochondria is crucial for 
cellular survival. The characterization of metaxin provides an opportunity to 
elucidate similarities and possible differences in the mechanisms of protein 
import between fungi and mammals and in the phenotypes of fungi and mammals 
lacking mitochondrial import receptors.