Effect of Mutations in Tom40 on Stability of the Translocase of the Outer Mitochondrial Membrane (TOM) Complex, Assembly of Tom40, and Import of Mitochondrial Preproteins

Abstract

Mitochondrial preproteins synthesized in the cytosol are imported through the mitochondrial outer membrane by the translocase of the outer mitochondrial membrane (TOM) complex. Tom40 is the major component of the complex and is essential for cell viability. We generated 21 different mutations in conserved regions of the Neurospora crassa Tom40 protein.The mutant genes were transformed into a tom40 null nucleus maintained in a sheltered heterokaryon, and 17 of the mutant genes gave rise to viable strains. All mutations reduced the efficiency of the altered Tom40 molecules to assemble into the TOM complex. Mitochondria isolated from seven of the mutant strains had defects for importing mitochondrial preproteins. Only one strain had a general import defect for all preproteins examined. Another mutation resulted in defects in the import of a matrix-destined preprotein and an outer membrane ␤-barrel protein, but import of the ADP/ATP carrier to the inner membrane was unaffected. Five strains showed deficiencies in the import of ␤-barrel proteins. The latter results suggest that the TOM complex distinguishes ␤-barrel proteins from other classes of preprotein during import. This supports the idea that the TOM complex plays an active role in the transfer of preproteins to subsequent translocases for insertion into the correct mitochondrial subcompartment.

Evidence Supporting the 19 β-Strand Model for Tom40 from Cysteine Scanning and Protease Site Accessibility Studies

“…Another study characterized several additional mutants involving deletion or Ala substitution of conserved Tom40 sequences (40). When examined in the context of the N. crassa Tom40 19-strand model, 14 of the mutants have deletions or Ala substitutions of residues affecting predicted ␤-strands (Fig.…”

“…To gain further insight into the possibility of maintaining Tom40 assembly and function when ␤ strands are interrupted, we re-examined previous work (19,40) on the effect of specific mutations on the assembly and function of NcTom40. These studies were completed prior to the appearance of the 19 ␤-strand three-dimensional structure for porin and the modeled Tom40 structure.…”

“…Thus, our results favor the modeling of Tom40 as a structure containing 19 ␤-strands. Previous studies on NcTom40 (19,40) that described the effects of mutations on its function, assembly, and stability have been re-examined in light of the current 19 ␤-strand model of the Tom40 structure.…”

Supramolecular Organization of the Respiratory Chain in Neurospora crassa Mitochondria

“…The mitochondrial import receptor Tom40 (Fig. 2, spot 42) was also identified as a subunit of the TOM holo or core complex (2,63,75,76,82) and found to comigrate with monomeric complex IV and heterooligomeric NAD ϩ -dependent isocitrate dehydrogenase (Fig. 2, spots 40 and 41).…”

Mitochondrial Protein Synthesis, Import, and Assembly

“…The N and C termini initially remain on the outside while internal regions containing recognition signals traverse the TOM complex as looped polypeptide chains through the Tom40 pore in the outer membrane Neupert and Herrmann 2007;Chacinska et al 2009). The Tom40 pore appears to have specific interactions with different imported proteins and thus may also play an active role in substrate recognition (Gabriel et al 2003;Sherman et al 2006).…”