The role of plant mitochondrial outer membrane proteins in the process of 
preprotein import was investigated, as some of the principal components 
characterized in yeast have been shown to be absent or evolutionarily distinct 
in plants. Three outer membrane proteins of Arabidopsis thaliana mitochondria 
were studied: TOM20 (translocase of the outer mitochondrial membrane), METAXIN, 
and mtOM64 (outer mitochondrial membrane protein of 64 kD). A single functional 
Arabidopsis TOM20 gene is sufficient to produce a normal multisubunit 
translocase of the outer membrane complex. Simultaneous inactivation of two of 
the three TOM20 genes changed the rate of import for some precursor proteins, 
revealing limited isoform subfunctionalization. Inactivation of all three TOM20 
genes resulted in severely reduced rates of import for some but not all 
precursor proteins. The outer membrane protein METAXIN was characterized to play 
a role in the import of mitochondrial precursor proteins and likely plays a role 
in the assembly of beta-barrel proteins into the outer membrane. An outer 
mitochondrial membrane protein of 64 kD (mtOM64) with high sequence similarity 
to a chloroplast import receptor was shown to interact with a variety of 
precursor proteins. All three proteins have domains exposed to the cytosol and 
interacted with a variety of precursor proteins, as determined by pull-down and 
yeast two-hybrid interaction assays. Furthermore, inactivation of one resulted 
in protein abundance changes in the others, suggesting functional redundancy. 
Thus, it is proposed that all three components directly interact with precursor 
proteins to participate in early stages of mitochondrial protein import.