Dissection of the Mitochondrial Import and Assembly Pathway for Human Tom40

Abstract

Tom40 is the channel-forming subunit of the translocase of the mitochondrial outer membrane (TOM complex), essential for protein import into mitochondria. Tom40 is synthesized in the cytosol and contains information for its mitochondrial targeting and assembly. A number of stable import intermediates have been identified for Tom40 precursors in fungi, the first being an association with the sorting and assembly machinery (SAM) of the outer membrane. By examining the import pathway of human Tom40, we have been able to elucidate additional features in its import. We identify that Hsp90 is involved in delivery of the Tom40 precursor to mitochondria in an ATP-dependent manner. The precursor then forms its first stable intermediate with the outer face of the TOM complex before its membrane integration and assembly. Deletion of an evolutionary conserved region within Tom40 disrupts the TOM complex intermediate and causes it to stall at a new complex in the intermembrane space that we identify to be the mammalian SAM. Unlike its fungal counterparts, the human Tom40 precursor is not found stably arrested at a SAM intermediate. Nevertheless, we show that Tom40 assembly is reduced in mitochondria depleted of human Sam50. These findings are discussed in context with current models from fungal studies.

Neisserial Omp85 Protein Is Selectively Recognized and Assembled into Functional Complexes in the Outer Membrane of Human Mitochondria

“…Mitochondrial isolation and carbonate extraction using 100 mM Na 2 CO 3 , pH 11.5, were performed as described previously (5,11,22). For opening of the OMM, freshly prepared mitochondria were incubated in isotonic (250 mM sucrose, 1 mM EDTA, 10 mM Tris, pH 7.6) or hypotonic (1 mM EDTA, 10 mM Tris, pH 7.6) buffer.…”

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

“…The observation that a single amino acid change in the primary sequence of Tom40 affected only transfer of preproteins to the TIM23 complex, but not to the TIM22 complex, suggested that Tom40 plays an active role in sorting proteins to the next mitochondrial translocase (47). Furthermore, a series of conserved residues near the N and C termini of the protein have been shown to be required for Tom40 assembly but not for receptor recognition (30,33,48). In this study we describe the effects of changing several regions containing conserved residues of Tom40 on the assembly, stability, and function of the TOM complex.…”

“…The protein is essential for viability in yeast and Neurospora crassa (29,30). Based on computer analysis, Tom40 is predicted to exist as a ␤-barrel within the mitochondrial outer membrane (31)(32)(33)(34). However, computer predictions differ regarding the number and position of the ␤-strands.…”

“…Further association with Tom6, Tom7, Tom22, and additional molecules of Tom40 give rise to the fully assembled 400-kDa TOM core complex. This pathway of assembly first described in fungal systems seems to be generally conserved in human mitochondria (33). Two additional proteins, Mim1/Tom13 (44,45) and Mdm10 (46), play a role in assembly of Tom40 following interaction with the TOB/SAM complex.…”

Alternative Splicing Gives Rise to Different Isoforms of the Neurospora crassa Tob55 Protein That Vary in Their Ability to Insert β-Barrel Proteins Into the Outer Mitochondrial Membrane

“…Homologs of yeast Tob55 are found in virtually all eukaryotes and in almost all gram-negative bacteria (Paschen et al 2003;Gentle et al 2004;Dolezal et al 2006). The group includes the bacterial Omp85/YaeT (Voulhoux et al 2003;Wu et al 2005), the plastid Toc75 (Eckart et al 2002), and the mammalian Tob55/Sam50 proteins (Humphries et al 2005). The N-terminal domain of Tob55 has been shown to recognize precursors of b-barrel proteins.…”