Evidence supporting the 19 ß-strand model for Tom40 from cysteine scanning and protease site accessibility studies.

Most proteins found in mitochondria are translated in the cytosol and enter the organelle via the TOM complex (translocase of the outer mitochondrial membrane). Tom40 is the pore forming component of the complex. Although the three-dimensional structure of Tom40 has not been determined, the structure of porin, a related protein, has been shown to be a ß-barrel containing 19 membrane spanning ß-strands and an N-terminal α-helical region. The evolutionary relationship between the two proteins has allowed modeling of Tom40 into a similar structure by several laboratories. However, it has been suggested that the 19-strand porin structure does not represent the native form of the protein. If true, modeling of Tom40 based on the porin structure would also be invalid. We have used substituted cysteine accessibility mapping to identify several potential ß-strands in the Tom40 protein in isolated mitochondria. These data, together with protease accessibility studies, support the 19 ß-strand model for Tom40 with the C-terminal end of the protein localized to the intermembrane space.

Analysis of mutations in Neurospora crassa ERMES components reveals specific functions related to ß-barrel protein assembly and maintenance of mitochondrial morphology.

The endoplasmic reticulum mitochondria encounter structure (ERMES) tethers the er to mitochondria and contains four structural components: Mmm1, Mdm12, Mdm10, and Mmm2 (Mdm34). The Gem1 protein may play a role in regulating ERMES function. Saccharomyces cerevisiae and Neurospora crassa strains lacking any of Mmm1, Mdm12, or Mdm10 are known to show a variety of phenotypic defects including altered mitochondrial morphology and defects in the assembly of ß-barrel proteins into the mitochondrial outer membrane. Here we examine ERMES complex components in N. crassa and show that Mmm1 is an ER membrane protein containing a Cys residue near its N-terminus that is conserved in the class Sordariomycetes. The residue occurs in the ER-lumen domain of the protein and is involved in the formation of disulphide bonds that give rise to Mmm1 dimers. Dimer formation is required for efficient assembly of Tom40 into the TOM complex. However, no effects are seen on porin assembly or mitochondrial morphology. This demonstrates a specificity of function and suggests a direct role for Mmm1 in Tom40 assembly. Mutation of a highly conserved region in the cytosolic domain of Mmm1 results in moderate defects in Tom40 and porin assembly, as well as a slight morphological phenotype. Previous reports have not examined the role of Mmm2 with respect to mitochondrial protein import and assembly. Here we show that absence of Mmm2 affects assembly of ß-barrel proteins and that lack of any ERMES structural component results in defects in Tom22 assembly. Loss of N. crassa Gem1 has no effect on the assembly of these proteins but does affect mitochondrial morphology.

Characterization of the insertase for ß-barrel proteins of the outer mitochondrial membrane.

The TOB-SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of ß-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55-Sam50, Tob38-Sam35, and Tob37-Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB-SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.

The Neurospora crassa TOB complex: analysis of the topology and function of Tob38 and Tob37.

The TOB or SAM complex is responsible for assembling several proteins into the mitochondrial outer membrane, including all ß-barrel proteins. We have identified several forms of the complex in Neurospora crassa. One form contains Tob55, Tob38, and Tob37; another contains these three subunits plus the Mdm10 protein; while additional complexes contain only Tob55. As previously shown for Tob55, both Tob37 and Tob38 are essential for viability of the organism. Mitochondria deficient in Tob37 or Tob38 have reduced ability to assemble ß-barrel proteins. The function of two hydrophobic domains in the C-terminal region of the Tob37 protein was investigated. Mutant Tob37 proteins lacking either or both of these regions are able to restore viability to cells lacking the protein. One of the domains was found to anchor the protein to the outer mitochondrial membrane but was not necessary for targeting or association of the protein with mitochondria. Examination of the import properties of mitochondria containing Tob37 with deletions of the hydrophobic domains reveals that the topology of Tob37 may be important for interactions between specific classes of ß-barrel precursors and the TOB complex.

Roles of the Mdm10, Tom7, Mdm12, and Mmm1 proteins in the assembly of mitochondrial outer membrane proteins in Neurospora crassa.

The Mdm10, Mdm12, and Mmm1 proteins have been implicated in several mitochondrial functions including mitochondrial distribution and morphology, assembly of beta-barrel proteins such as Tom40 and porin, association of mitochondria and endoplasmic reticulum, and maintaining lipid composition of mitochondrial membranes. Here we show that loss of any of these three proteins in Neurospora crassa results in the formation of large mitochondrial tubules and reduces the assembly of porin and Tom40 into the outer membrane. We have also investigated the relationship of Mdm10 and Tom7 in the biogenesis of beta-barrel proteins. Previous work showed that mitochondria lacking Tom7 assemble Tom40 more efficiently, and porin less efficiently, than wild-type mitochondria. Analysis of mdm10 and tom7 single and double mutants, has demonstrated that the effects of the two mutations are additive. Loss of Tom7 partially compensates for the decrease in Tom40 assembly resulting from loss of Mdm10, whereas porin assembly is more severely reduced in the double mutant than in either single mutant. The additive effects observed in the double mutant suggest that different steps in beta-barrel assembly are affected in the individual mutants. Many aspects of Tom7 and Mdm10 function in N. crassa are different from those of their homologues in Saccharomyces cerevisiae.