The essential mitochondrial protein Erv1 cooperates with Mia40 in biogenesis of intermembrane space proteins.

The proteins of the mitochondrial intermembrane space (IMS) are encoded by nuclear genes and synthesized on cytosolic ribosomes. While some IMS proteins are imported by the classical presequence pathway that involves the membrane potential deltapsi across the inner mitochondrial membrane and proteolytic processing to release the mature protein to the IMS, the import of numerous small IMS proteins is independent of a deltapsi and does not include proteolytic processing. The biogenesis of small IMS proteins requires an essential mitochondrial IMS import and assembly protein, termed Mia40. Here, we show that Erv1, a further essential IMS protein that has been reported to function as a sulfhydryl oxidase and participate in biogenesis of Fe/S proteins, is also required for the biogenesis of small IMS proteins. We generated a temperature-sensitive yeast mutant of Erv1 and observed a strong reduction of the levels of small IMS proteins upon shift of the cells to non-permissive temperature. Isolated erv1-2 mitochondria were selectively impaired in import of small IMS proteins while protein import pathways to other mitochondrial subcompartments were not affected. Small IMS precursor proteins remained associated with Mia40 in erv1-2 mitochondria and were not assembled into mature oligomeric complexes. Moreover, Erv1 associated with Mia40 in a reductant-sensitive manner. We conclude that two essential proteins, Mia40 and Erv1, cooperate in the assembly pathway of small proteins of the mitochondrial IMS.

Influence of redox-active compounds and PXR-activators on human MRP1 and MRP2 gene expression.

In the present study, we investigated the inducibility of the drug conjugate transporter genes MRP1 and MRP2 by redox-active compounds such as tertiary butylated hydroquinone (tBHQ) and quercetin and by chemicals known to activate the pregnane X receptor (PXR) such as rifampicin and clotrimazol and by the metalloid compound arsenite. The human MRP2 gene was found to be inducible in HepG2 cells by rifampicin, clotrimazol, arsenite and tBHQ. As MRP1 expression is extremely low in HepG2 cells, its inducibility was studied in MCF-7 cells. However, only tBHQ and quercetin acted as inducers, but not the other compounds investigated. Reporter gene assays demonstrated that proximal promoter regions of the genes contribute to the induction by tBHQ, quercetin (MRP1) and clotrimazol (MRP2). However, the deletion of binding sites supposed to mediate the induction process (a PXR-binding element-like sequence for the clotrimazol effect and an ARE (antioxidative response element) for the tBHQ/quercetin effect) did not result in a significant decrease in the induction factor indicating that other parts of the promoter are probably involved in the induction process. In summary, expression of both genes can be up-regulated by redox-active compounds, while the other compounds tested induced only MRP2 but not MRP1 expression.

Biogenesis of mitochondrial ß-barrel proteins: the POTRA domain is involved in precursor release from the SAM complex.

The mitochondrial outer membrane contains proteinaceous machineries for the translocation of precursor proteins. The sorting and assembly machinery (SAM) is required for the insertion of ß-barrel proteins into the outer membrane. Sam50 is the channel-forming core subunit of the SAM complex and belongs to the BamA/Sam50/Toc75 family of proteins that have been conserved from Gram-negative bacteria to mitochondria and chloroplasts. These proteins contain one or more N-terminal polypeptide transport-associated (POTRA) domains. POTRA domains can bind precursor proteins, however, different views exist on the role of POTRA domains in the biogenesis of ß-barrel proteins. It has been suggested that the single POTRA domain of mitochondrial Sam50 plays a receptor-like function at the SAM complex. We established a system to monitor the interaction of chemical amounts of ß-barrel precursor proteins with the SAM complex of wild-type and mutant yeast in organello. We report that the SAM complex lacking the POTRA domain of Sam50 efficiently binds ß-barrel precursors, but is impaired in the release of the precursors. These results indicate the POTRA domain of Sam50 is not essential for recognition of ß-barrel precursors but functions in a subsequent step to promote the release of precursor proteins from the SAM complex.

Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.

The morphology proteins Mdm12/Mmm1 function in the major beta-barrel assembly pathway of mitochondria.

The beta-barrel proteins of mitochondria are synthesized on cytosolic ribosomes. The proteins are imported by the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been assumed that the SAM(core) complex with the subunits Sam35, Sam37 and Sam50 represents the last import stage common to all beta-barrel proteins, followed by splitting in a Tom40-specific route and a route for other beta-barrel proteins. We have identified new components of the beta-barrel assembly machinery and show that the major beta-barrel pathway extends beyond SAM(core). Mdm12/Mmm1 function after SAM(core) yet before splitting of the major pathway. Mdm12/Mmm1 have been known for their role in maintenance of mitochondrial morphology but we reveal assembly of beta-barrel proteins as their primary function. Moreover, Mdm10, which functions in the Tom40-specific route, can associate with SAM(core) as well as Mdm12/Mmm1 to form distinct assembly complexes, indicating a dynamic exchange between the machineries governing mitochondrial beta-barrel assembly. We conclude that assembly of mitochondrial beta-barrel proteins represents a major function of the morphology proteins Mdm12/Mmm1.

Biogenesis of the protein import channel Tom40 of the mitochondrial outer membrane: intermembrane space components are involved in an early stage of the assembly pathway.

Tom40 forms the central channel of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The precursor of Tom40 is encoded in the nucleus, synthesized in the cytosol, and imported into mitochondria via a multi-step assembly pathway that involves the mature TOM complex and the sorting and assembly machinery of the outer membrane (SAM complex). We report that opening of the mitochondrial intermembrane space by swelling blocks the assembly pathway of the beta-barrel protein Tom40. Mitochondria with defects in small Tim proteins of the intermembrane space are impaired in the Tom40 assembly pathway. Swelling as well as defects in the small Tim proteins inhibit an early stage of the Tom40 import pathway that is needed for formation of a Tom40-SAM intermediate. We propose that the biogenesis pathway of beta-barrel proteins of the outer mitochondrial membrane not only requires TOM and SAM components, but also involves components of the intermembrane space.

Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins.

Mitochondria import nuclear-encoded precursor proteins to four different subcompartments. Specific import machineries have been identified that direct the precursor proteins to the mitochondrial outer membrane, inner membrane or matrix, respectively. However, a machinery dedicated to the import of mitochondrial intermembrane space (IMS) proteins has not been found so far. We have identified the essential IMS protein Mia40 (encoded by the Saccharomyces cerevisiae open reading frame YKL195w). Mitochondria with a mutant form of Mia40 are selectively inhibited in the import of several small IMS proteins, including the essential proteins Tim9 and Tim10. The import of proteins to the other mitochondrial subcompartments does not depend on functional Mia40. The binding of small Tim proteins to Mia40 is crucial for their transport across the outer membrane and represents an initial step in their assembly into IMS complexes. We conclude that Mia40 is a central component of the protein import and assembly machinery of the mitochondrial IMS.