Roles of the Mdm10, Tom7, Mdm12, and Mmm1 Proteins in the Assembly of Mitochondrial Outer Membrane Proteins in Neurospora crassa

Abstract

The Mdm10, Mdm12, and Mmm1 proteins have been implicated in several mitochondrial functions including mitochondrial distribution and morphology, assembly of ␤-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 ␤-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 ␤-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.

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

“…For SCAM analysis the cells were ground using a mortar and pestle in the presence of sand and PBSSP isolation buffer (phosphate-buffered salts, sucrose, phenylmethylsulfonyl fluoride (PMSF): 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 , pH 7.4, 0.25 M sucrose, 1 mM PMSF). Mitochondria were isolated by differential centrifugation as described previously (43,44). For all other experiments, mitochondria were isolated using buffers described previously (43).…”

Trypanosomal TAC40 constitutes a novel subclass of mitochondrial β-barrel proteins specialized in mitochondrial genome inheritance

“…The ERMES has also been suggested to connect to cytosolic actin fibers that mediate the movement of mitochondria to the bud of dividing yeast cells (14,18,19). Besides its role in mitochondrial inheritance, the ERMES has been implicated in maintenance of mitochondrial morphology and in phospholipid and calcium exchange as well as in the assembly of the protein translocase of the mitochondrial OM (TOM) (20,21). Some of the proposed ERMES functions are controversial and there is evidence that some of them might be due to secondary effects caused by the drastically altered mitochondrial morphology (22).…”

“…Moreover, a fraction of the protein is associated with the SAM, where it mediates assembly of the TOM complex (21,29). In T. brucei the shape of the mitochondrion shows marked differences between the procyclic and the bloodstream form.…”

The ubiquitous and ancient ER membrane protein complex (EMC): tether or not?

“…The ER-mitochondria encounter structure (ERMES), also involved in ER-mitochondria tethering, is a multifunctional protein complex implicated in both lipid transfer and mitochondrial outer membrane protein assembly ( AhYoung et al , 2015; Kornmann et al , 2009; Meisinger et al , 2006, Meisinger et al , 2007; Wideman et al , 2013; Wideman et al , 2010). However, ERMES as an ER-mitochondria tether is limited to a subset of eukaryote taxa ( Wideman et al , 2013), suggesting that a universal ER-mitochondria tethering complex might exist.…”