The preprotein translocase of the outer mitochondrial membrane (TOM) consists of a central beta-barrel channel, Tom40, and six proteins with alpha-helical transmembrane segments. The precursor of Tom40 is imported from the cytosol by a pre-existing TOM complex and inserted into the outer membrane by the sorting and assembly machinery (SAM). Tom40 then assembles with alpha-helical Tom proteins to the mature TOM complex. The outer membrane protein Mim1 promotes membrane insertion of several alpha-helical Tom proteins but also affects the biogenesis of Tom40 by an unknown mechanism. We have identified a novel intermediate in the assembly pathway of Tom40, revealing a two-stage interaction of the precursor with the SAM complex. The second SAM stage represents assembly of Tom5 with the precursor of Tom40. Mim1-deficient mitochondria accumulate Tom40 at the first SAM stage like Tom5-deficient mitochondria. Tom5 promotes formation of the second SAM stage and thus suppresses the Tom40 assembly defect of mim1Delta mitochondria. We conclude that the assembly of newly imported Tom40 is directly initiated at the SAM complex by its association with Tom5. The involvement of Mim1 in Tom40 biogenesis can be largely attributed to its role in import of Tom5.
- Membrane Proteins/genetics/*metabolism Mitochondria/*metabolism Mitochondrial Membrane Transport Proteins/genetics/*metabolism Multiprotein Complexes/*metabolism Saccharomyces cerevisiae/cytology/metabolism Saccharomyces cerevisiae Proteins/genetics/*metabolism