The outer membrane form of the mitochondrial protein Mcr1 follows a TOM-independent membrane insertion pathway

The yeast gene MCR1 encodes two isoforms of the mitochondrial NADH-cytochrome b5 reductase. One form is embedded in the outer membrane whereas the other is located in the intermembrane space (IMS). In the present work we investigated the biogenesis of the outer membrane form. We demonstrate that while the IMS form crosses the outer membrane via the translocase of the outer mitochondrial membrane (TOM) complex, the other form is integrated into the outer membrane by a process that does not require any of the known import components at the outer membrane. Thus, the import pathways of the two forms diverge in a stage before the encounter with the TOM complex and their mechanism of biogenesis represents a unique example how to achieve dual localization within one organelle.     

Preprotein transport machineries of yeast mitochondrial outer membrane are not required for Bax-induced release of intermembrane space proteins

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.     

Cryo-electron microscopy structure of a yeast mitochondrial preprotein translocase

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for proteins imported into mitochondria. We determined the structure of the native, unstained ∼ 550-kDa core-Tom20 complex from Saccharomycescerevisiae by cryo-electron microscopy at 18-Å resolution. The complex is triangular, measuring 145 Å on edge, and has near-3-fold symmetry. Its bulk is made up of three globular ∼ 50-Å domains. Three elliptical pores on the c-face merge into one central ∼ 70-Å cavity with a cage-like assembly on the opposite t-face. Nitrilotriacetic acid-gold labeling indicates that three Tom22 subunits in the TOM complex are located at the perimeter of the complex near the interface of the globular domains. We assign Tom22, which controls complex assembly, to three peripheral protrusions on the c-face, while the Tom20 subunit is tentatively assigned to the central protrusion on this surface. Based on our three-dimensional map, we propose a model of transient interactions and functional dynamics of the TOM assembly.     

Novel mitochondrial intermembrane space proteins as substrates of the MIA import pathway

Mitochondria consist of four compartments, the outer membrane, intermembrane space (IMS), inner membrane and the matrix. Most mitochondrial proteins are synthesized as precursors in the cytosol and have to be imported into these compartments. While the protein import machineries of the outer membrane, inner membrane and matrix have been investigated in detail, a specific mitochondrial machinery for import and assembly of IMS proteins, termed MIA, was identified only recently. To date, only a very small number of substrate proteins of the MIA pathway have been identified. The substrates contain characteristic cysteine motifs, either a twin Cx(3)C or a twin Cx(9)C motif. The largest MIA substrates known possess a molecular mass of 11 kDa, implying that this new import pathway has a very small size limit. Here, we have compiled a list of Saccharomyces cerevisiae proteins with a twin Cx(9)C motif and identified three IMS proteins that were previously localized to incorrect cellular compartments by tagging approaches. Mdm35, Mic14 (YDR031w) and Mic17 (YMR002w) require the two essential subunits, Mia40 and Erv1, of the MIA machinery for their localization in the mitochondrial IMS. With a molecular mass of 14 kDa and 17 kDa, respectively, Mic14 and Mic17 are larger than the known MIA substrates. Remarkably, the precursor of Erv1 itself is imported via the MIA pathway. As Erv1 has a molecular mass of 22 kDa and a twin Cx(2)C motif, this study demonstrates that the MIA pathway can transport substrates that are twice as large as the substrates known to date and is not limited to proteins with twin Cx(3)C or Cx(9)C motifs. However, tagging of MIA substrates can interfere with their subcellular localization, indicating that the proper localization of mitochondrial IMS proteins requires the characterization of the authentic untagged proteins.     

Distinctive biochemistry in the trypanosome mitochondrial intermembrane space suggests a model for stepwise evolution of the MIA pathway for import of cysteine-rich proteins

Mia40-dependent disulphide bond exchange is used by animals, yeast, and probably plants for import of small, cysteine-rich proteins into the mitochondrial intermembrane space (IMS). During import, electrons are transferred from the imported substrate to Mia40 then, via the sulphydryl oxidase Erv1, into the respiratory chain. Curiously, however, there are protozoa which contain substrates for Mia40-dependent import, but lack Mia40. There are also organisms where Erv1 is present in the absence of respiratory chain components. In accommodating these and other relevant observations pertaining to mitochondrial cell biology, we hypothesise that the ancestral IMS import pathway for disulphide-bonded proteins required only Erv1 (but not Mia40) and identify parasites in which O(2) is the likely physiological oxidant for Erv1.     

A Tag at the Carboxy Terminus Prevents Membrane Integration of VDAC1 in Mammalian Mitochondria

β-Barrel proteins are found in the outer membranes of bacteria, chloroplasts and mitochondria. The evolutionary conserved sorting and assembly machinery (SAM complex) assembles mitochondrial β-barrel proteins, such as voltage-dependent anion-selective channel 1 (VDAC1), into complexes in the outer membrane by recognizing a sorting β-signal in the carboxy-terminal part of the protein. Here we show that in mammalian mitochondria, masking of the C-terminus of β-barrel proteins by a tag leads to accumulation of soluble misassembled protein in the intermembrane space, which causes mitochondrial fragmentation and loss of membrane potential. A similar phenotype is observed if the β-signal is shortened, removed or when the conserved hydrophobic residues in the β-signal are mutated. The length of the tag at the C-terminus is critical for the assembly of VDAC1, as well as the amino acid residues at positions 130, 222, 225 and 251 of the protein. We propose that if the recognition of the β-signal or the folding of the β-barrel proteins is inhibited, the nonassembled protein will accumulate in the intermembrane space, aggregate and damage mitochondria. This effect offers easy tools for studying the requirements for the membrane assembly of β-barrel proteins, but also advises caution when interpreting the outcome of the β-barrel protein overexpression experiments.