Structural requirements of Tom40 for assembly into preexisting TOM complexes of mitochondria

Tom40 is the major subunit of the translocase of the outer mitochondrial me mbrane (the TOM complex). To study the assembly pathway of Tom40, we have f ollowed the integration of the protein into the TOM complex in vitro and in vivo using wild-type and altered versions of the Neurospora crassa Tom40 p rotein. Upon import into isolated mitochondria, Tom40 precursor proteins la cking the first 20 or the first 40 amino acid residues were assembled as th e wild-type protein. In contrast, a Tom40 precursor lacking residues 41 to 60, which contains a highly conserved region of the protein, was arrested a t an intermediate stage of assembly. We constructed mutant versions of Tom4 0 affecting this region and transformed the genes into a sheltered heteroka ryon containing a tom40 null nucleus. Homokaryotic strains expressing the m utant Tom40 proteins had growth rate defects and were deficient in their ab ility to form conidia. Analysis of the TOM complex in these strains by blue native gel electrophoresis revealed alterations in electrophoretic mobilit y and a tendency to lose Tom40 subunits from the complex. Thus, both in vit ro and in vivo studies implicate residues 41 to 60 as containing a sequence required for proper assembly/stability of Tom40 into the TOM complex. Fina lly, we found that TOM complexes in the mitochondrial outer membrane were c apable of exchanging subunits in vitro. A model is proposed for the integra tion of Tom40 subunits into the TOM complex.