Alternative splicing gives rise to different isoforms of the Neurospora crassa Tob55 protein that vary in their ability to insert beta-barrel proteins into the outer mitochondrial membrane.

Tob55 is the major component of the TOB complex, which is found in the outer membrane of mitochondria. A sheltered knockout of the tob55 gene was developed in Neurospora crassa. When grown under conditions that reduce the levels of the Tob55 protein, the strain exhibited a reduced growth rate and mitochondria isolated from these cells were deficient in their ability to import beta-barrel proteins. Surprisingly, Western blots of wild-type mitochondrial proteins revealed two bands for Tob55 that differed by approximately 4 kDa in their apparent molecular masses. Sequence analysis of cDNAs revealed that the tob55 mRNA is alternatively spliced and encodes three isoforms of the protein, which are predicted to contain 521, 516, or 483 amino acid residues. Mass spectrometry of proteins isolated from purified outer membrane vesicles confirmed the existence of each isoform in mitochondria. Strains that expressed each isoform of the protein individually were constructed. When cells expressing only the longest form of the protein were grown at elevated temperature, their growth rate was reduced and mitochondria isolated from these cells were deficient in their ability to assembly beta-barrel proteins.

The Tim8-Tim13 complex of Neurospora crassa functions in the assembly of proteins into both mitochondrial membranes.

The Tim8 and Tim13 proteins in yeast are known to exist in the mitochondrial intermembrane space and to form a hetero-oligomeric complex involved in the import of the mitochondrial inner membrane protein Tim23, the central component of the TIM23 translocase. Here, we have isolated tim8 and tim13 mutants in Neurospora crassa and have shown that mitochondria lacking the Tim8-Tim13 complex were deficient in the import of the outer membrane beta-barrel proteins Tom40 and porin. Cross-linking studies showed that the Tom40 precursor contacts the Tim8-Tim13 complex. The complex is involved at an early point in the Tom40 assembly pathway because cross-links can only be detected during the initial stages of Tom40 import. In mitochondria lacking the Tim8-Tim13 complex, the Tom40 precursor appears in a previously characterized early intermediate of Tom40 assembly more slowly than in wild type mitochondria. Thus, our data suggest a model in which one of the first steps in Tom40 assembly may be interaction with the Tim8-Tim13 complex. As in yeast, the N. crassa Tim23 precursor was imported inefficiently into mitochondria lacking the Tim8-Tim13 complex when the membrane potential was reduced. Tim23 import intermediates could also be cross-linked to the complex, suggesting a dual role for the Tim8-Tim13 intermembrane space complex in the import of proteins found in both the outer and inner mitochondrial membranes.

Tim50, a novel component of the TIM23 preprotein translocase of mitochondria.

The preprotein translocase of the inner membrane of mitochondria (TIM23 complex) is the main entry gate for proteins of the matrix and the inner membrane. We isolated the TIM23 complex of Neurospora crassa. Besides Tim23 and Tim17, it contained a novel component, referred to as Tim50. Tim50 spans the inner membrane with a single transmembrane segment and exposes a large hydrophilic domain in the intermembrane space. Tim50 is essential for viability of yeast. Mitochondria from cells depleted of Tim50 displayed strongly reduced import kinetics of preproteins using the TIM23 complex. Tim50 could be cross-linked to preproteins that were halted at the level of the translocase of the outer membrane (TOM complex) or spanning both TOM and TIM23 complexes. We suggest that Tim50 plays a crucial role in the transfer of preproteins from the TOM complex to the TIM23 complex through the intermembrane space.