The Mitochondrial Import Complex MIM Functions as Main Translocase for α-Helical Outer Membrane Proteins.

The mitochondrial outer membrane contains integral proteins with α-helical membrane anchors or a transmembrane ß-barrel. The translocase of the outer membrane (TOM) cooperates with the sorting and assembly machinery (SAM) in the import of ß-barrel proteins, whereas the mitochondrial import (MIM) complex inserts precursors of multi-spanning α-helical proteins. Single-spanning proteins constitute more than half of the integral outer membrane proteins; however, their biogenesis is poorly understood. We report that the yeast MIM complex promotes the insertion of proteins with N-terminal (signal-anchored) or C-terminal (tail-anchored) membrane anchors. The MIM complex exists in three dynamic populations. MIM interacts with TOM to accept precursor proteins from the receptor Tom70. Free MIM complexes insert single-spanning proteins that are imported in a Tom70-independent manner. Finally, coupling of MIM and SAM promotes early assembly steps of TOM subunits. We conclude that the MIM complex is a major and versatile protein translocase of the mitochondrial outer membrane.

Microenvironmental stromal cells abrogate NF-κB inhibitor-induced apoptosis in chronic lymphocytic leukemia.

Nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) is known to play an important role in the pathogenesis of chronic lymphocytic leukemia (CLL). Several NF-κB inhibitors were shown to successfully induce apoptosis of CLL cells in vitro Since the microenvironment is known to be crucial for the survival of CLL cells, herein, we tested whether NF-κB inhibition may still induce apoptosis in these leukemic cells in the presence of protective stromal interaction. We used the specific NF-κB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ). Microenvironmental support was mimicked by co-culturing CLL cells with bone marrow-derived stromal cell lines (HS-5 and M2-10B4). NF-κB inhibition by DHMEQ in CLL cells could be confirmed in both the monoculture and co-culture setting. In line with previous reports, NF-κB inhibition induced apoptosis in the monoculture setting by activating the intrinsic apoptotic pathway resulting in poly (ADP-ribose) polymerase (PARP)-cleavage; however, it was unable to induce apoptosis in leukemic cells co-cultured with stromal cells. Similarly, small interfering ribonucleic acid (siRNA)-mediated RELA downregulation induced apoptosis of CLL cells cultured alone, but not in the presence of supportive stromal cells. B-cell activating factor (BAFF) was identified as a microenvironmental messenger potentially protecting the leukemic cells from NF-κB inhibition-induced apoptosis. Finally, we show improved sensitivity of stroma-supported CLL cells to NF-κB inhibition when combining the NF-κB inhibitor with the SYK inhibitor R406 or the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib, agents known to inhibit the stroma-leukemia crosstalk. We conclude that NF-κB inhibitors are not promising as monotherapies in CLL, but may represent attractive therapeutic partners for ibrutinib and R406.

Coupling of mitochondrial import and export translocases by receptor-mediated supercomplex formation.

The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of ß-barrel proteins use both translocases-TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the ß-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring ß-barrel formation in vivo and in organello and demonstrated that the ß-barrel was formed and membrane inserted while the precursor was bound to SAM. ß-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling.

Biogenesis of mitochondria: dual role of Tom7 in modulating assembly of the preprotein translocase of the outer membrane.

Biogenesis of the translocase of the outer mitochondrial membrane (TOM complex) involves the assembly of the central ß-barrel forming protein Tom40 with six different subunits that are embedded in the membrane via α-helical transmembrane segments. The sorting and assembly machinery (SAM complex) of the outer membrane plays a central role in this process. The SAM complex mediates the membrane integration of ß-barrel precursor proteins including Tom40. The small Tom proteins Tom5 and Tom6 associate with the precursor of Tom40 at the SAM complex at an early stage of the assembly process and play a stimulatory role in the formation of the mature TOM complex. A fraction of the SAM components interacts with the outer membrane protein mitochondrial distribution and morphology protein 10 (Mdm10) to form the SAM-Mdm10 machinery; however, different views exist on the function of the SAM-Mdm10 complex. We report here that the third small Tom protein, Tom7, plays an inhibitory role at two distinct steps in the biogenesis of the TOM complex. First, Tom7 plays an antagonistic role to Tom5 and Tom6 at the early stage of Tom40 assembly at the SAM complex. Second, Tom7 interacts with Mdm10 that is not bound to the SAM complex, and thus promotes dissociation of the SAM-Mdm10 complex. Since the SAM-Mdm10 complex is required for the biogenesis of Tom22, Tom7 delays the assembly of Tom22 with Tom40 at a late stage of assembly of the TOM complex. Thus, Tom7 modulates the biogenesis of topologically different proteins, the ß-barrel forming protein Tom40 and Tom22 that contains a transmembrane α-helix.

Assembly of the mitochondrial protein import channel: role of Tom5 in two-stage interaction of Tom40 with the SAM complex.

The preprotein translocase of the outer mitochondrial membrane (TOM) consists of a central ß-barrel channel, Tom40, and six proteins with α-helical transmembrane segments. The precursor of Tom40 is imported from the cytosol by a pre-existing TOM complex and inserted into the outer membrane by the sorting and assembly machinery (SAM). Tom40 then assembles with α-helical Tom proteins to the mature TOM complex. The outer membrane protein Mim1 promotes membrane insertion of several α-helical Tom proteins but also affects the biogenesis of Tom40 by an unknown mechanism. We have identified a novel intermediate in the assembly pathway of Tom40, revealing a two-stage interaction of the precursor with the SAM complex. The second SAM stage represents assembly of Tom5 with the precursor of Tom40. Mim1-deficient mitochondria accumulate Tom40 at the first SAM stage like Tom5-deficient mitochondria. Tom5 promotes formation of the second SAM stage and thus suppresses the Tom40 assembly defect of mim1Δ mitochondria. We conclude that the assembly of newly imported Tom40 is directly initiated at the SAM complex by its association with Tom5. The involvement of Mim1 in Tom40 biogenesis can be largely attributed to its role in import of Tom5.

Two modular forms of the mitochondrial sorting and assembly machinery are involved in biogenesis of alpha-helical outer membrane proteins.

The mitochondrial outer membrane contains two translocase machineries for precursor proteins--the translocase of the outer membrane (TOM complex) and the sorting and assembly machinery (SAM complex). The TOM complex functions as the main mitochondrial entry gate for nuclear-encoded proteins, whereas the SAM complex was identified according to its function in the biogenesis of beta-barrel proteins of the outer membrane. The SAM complex is required for the assembly of precursors of the TOM complex, including not only the beta-barrel protein Tom40 but also a subset of alpha-helical subunits. While the interaction of beta-barrel proteins with the SAM complex has been studied in detail, little is known about the interaction between the SAM complex and alpha-helical precursor proteins. We report that the SAM is not static but that the SAM core complex can associate with different partner proteins to form two large SAM complexes with different functions in the biogenesis of alpha-helical Tom proteins. We found that a subcomplex of TOM, Tom5-Tom40, associates with the SAM core complex to form a new large SAM complex. This SAM-Tom5/Tom40 complex binds the alpha-helical precursor of Tom6 after the precursor has been inserted into the outer membrane in an Mim1 (mitochondrial import protein 1)-dependent manner. The second large SAM complex, SAM-Mdm10 (mitochondrial distribution and morphology protein), binds the alpha-helical precursor of Tom22 and promotes its membrane integration. We suggest that the modular composition of the SAM complex provides a flexible platform to integrate the sorting pathways of different precursor proteins and to promote their assembly into oligomeric complexes.