Klaus-Peter Künkele

The Preprotein Translocation Channel of the Outer Membrane of Mitochondria

The preprotein translocase of the outer membrane of mitochondria (TOM complex) facilitates the recognition, insertion, and translocation of nuclear-encoded mitochondrial preproteins. We have purified the TOM complex from Neurospora crassa and analyzed its composition and functional properties. The TOM complex contains a cation-selective high-conductance channel. Upon reconstitution into liposomes, it mediates integration of proteins into and translocation across the lipid bilayer. TOM complex particles have a diameter of about 138 A, as revealed by electron microscopy and image analysis; they contain two or three centers of stain-filled openings, which we interpret as pores with an apparent diameter of about 20 A. We conclude that the structure reported here represents the protein-conducting channel of the mitochondrial outer membrane.

The Isolated Complex of the Translocase of the Outer Membrane of Mitochondria CHARACTERIZATION OF THE CATION-SELECTIVE AND VOLTAGE-GATED PREPROTEIN-CONDUCTING PORE

The complex of the translocase mitochondrial outer membrane (TOM), mediates recognition, unfolding, and translocation of preproteins. We have used a combination of biochemical and electrophysiological methods to study the properties of the preprotein-conducting pore of the purified TOM complex. The pore is cation-selective and voltage-gated. It shows three main conductance levels with characteristic slow and fast kinetics transitions to states of lower conductance following application of transmembrane voltages. These electrical properties distinguish it from the mitochondrial voltage-dependent anion channel (porin) and are identical to those of the previously described peptide-sensitive channel. Binding of antibodies to the C terminus of Tom40 on the intermembrane space side of the outer membrane modifies the channel properties and allows determination of the orientation of the channel within the lipid bilayer. Mitochondrial presequence peptides specifically interact with the pore and decrease the ion flow through the channel in a voltage-dependent manner. We propose that the presequence-induced closures of the pore are related to structural alterations of the TOM complex observed during the various stages of preprotein movement across the mitochondrial outer membrane.

Recognition of preproteins by the isolated TOM complex of mitochondria

A multisubunit complex in the mitochondrial outer membrane, the TOM complex, mediates targeting and membrane translocation of nuclear‐encoded preproteins. We have isolated the TOM holo complex, containing the preprotein receptor components Tom70 and Tom20, and the TOM core complex, which lacks these receptors. The interaction of recombinant mitochondrial preproteins with both types of soluble TOM complex was analyzed. Preproteins bound efficiently in a specific manner to the isolated complexes in the absence of chaperones and lipids in a bilayer structure. Using fluorescence correlation spectroscopy, a dissociation constant in the nanomolar range was determined. The affinity was lower when the preprotein was stabilized in its folded conformation. Following the initial binding, the presequence was transferred into the translocation pore in a step that required unfolding of the mature part of the preprotein. This translocation step was also mediated by protease‐treated TOM holo complex, which contains almost exclusively Tom40. Thus, the TOM core complex, consisting of Tom40, Tom22, Tom6 and Tom7, is a molecular machine that can recognize and partially translocate mitochondrial precursor proteins.

Dynamics of the TOM Complex of Mitochondria during Binding and Translocation of Preproteins

ABSTRACT Translocation of preproteins across the mitochondrial outer membrane is mediated by the TOM complex. This complex consists of receptor components for the initial contact with preproteins at the mitochondrial surface and membrane-embedded proteins which promote transport and form the translocation pore. In order to understand the interplay between the translocating preprotein and the constituents of the TOM complex, we analyzed the dynamics of the TOM complex ofNeurospora crassa and Saccharomyces cerevisiaemitochondria by following the structural alterations of the essential pore component Tom40 during the translocation of preproteins. Tom40 exists in a homo-oligomeric assembly and dynamically interacts with Tom6. The Tom40 assembly is influenced by a block of negatively charged amino acid residues in the cytosolic domain of Tom22, indicating a cross-talk between preprotein receptors and the translocation pore. Preprotein binding to specific sites on either side of the outer membrane (cis and trans sites) induces distinct structural alterations of Tom40. To a large extent, these changes are mediated by interaction with the mitochondrial targeting sequence. We propose that such targeting sequence-induced adaptations are a critical feature of translocases in order to facilitate the movement of preproteins across cellular membranes.

'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex.

MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of ‘sheltered disruption’, we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom‐22 gene and the other with a wild‐type allele. Homokaryons bearing the mom‐22 disruption could not be isolated, suggesting that mom‐22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22‐depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22‐depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22‐independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.

Preclinical evidence for nitrogen-containing bisphosphonate inhibition of farnesyl diphosphate (FPP) synthase in the kidney : Implications for renal safety

Bisphosphonates are potent inhibitors of osteoclast-mediated bone resorption and play an important role in the treatment of osteoporosis, metastatic bone disease, and Paget disease. However, nephrotoxicity has been reported with some bisphosphonates. Nitrogen-containing bisphosphonates directly inhibit farnesyl diphosphate (FPP) synthase activity (mevalonate pathway) and reduce protein prenylation leading to osteoclast cell death. The aim here was to elucidate if this inhibition also occurs in kidney cells and may directly account for nephrotoxicity. In an exploratory study in rats receiving zoledronate or ibandronate an approximate 2-fold increase in FPP synthase mRNA levels was observed in the kidney. The involvement of the mevalonate pathway was confirmed in subsequent in vitro studies with zoledronate, ibandronate, and pamidronate, using the non-nitrogen containing bisphosphonate clodronate as a comparator. In vitro changes in FPP synthase mRNA expression, enzyme activity, and levels of prenylated proteins were assessed. Using two cell lines (a rat normal kidney cell line, NRK-52E, and a human kidney proximal tubule cell line, HK-2), ibandronate and zoledronate were identified as most cytotoxic (EC50: 23/>1000 microM and 16/82 microM, respectively) and as the most potent inhibitors of FPP synthase (IC50; 1.6/7.4 microM and 0.5/0.7 microM, respectively). In both cell lines, inhibition of FPP synthase activity occurred prior to a decrease in levels of prenylated proteins followed by cytotoxicity. This further supports that the mechanism responsible for osteoclast inhibition (therapeutic effect) might also underlie the mechanism of nephrotoxicity.

Short‐hairpin‐RNA‐mediated silencing of fucosyltransferase 8 in Chinese‐hamster ovary cells for the production of antibodies with enhanced antibody immune effector function

Antibody‐producing Chinese‐hamster ovary cells (CHO‐DG44) were converted into cells producing antibodies with strongly enhanced ADCC (antibody‐dependent cellular cytotoxicity) by knocking down FuT8 (α‐1,6‐fucosyltransferase or fucosyltransferase 8) via constitutive expression of shRNA (short‐hairpin RNA) against FuT8. After the introduction of a FuT8 shRNA expression plasmid under the control of a U6 promoter, CHO‐DG44 clones with less than 5% residual FuT8 mRNA expression were isolated by selection for neomycin resistance, followed by low affinity nerve growth factor receptor enrichment and selection for LCA [Lens culinaris (culinary lentil) agglutinin] resistance. The CHO‐DG44 clones identified produced highly afucosylated anti‐[IGF‐1R (insulin‐like‐growth‐factor‐1 receptor)] antibodies (up to 88%) that exhibited considerably enhanced ADCC compared with anti‐IGF‐1R wild‐type antibodies produced by parental CHO cells. At the same time, antibody productivity was not significantly decreased. Analysis of stability showed that the clones obtained may be suitable for up‐scaling, since low residual levels of FuT8 mRNA and production of afucosylated antibodies were maintained for at least 4 weeks.

Protein Transport into and Across the Mitochondrial Outer Membrane: Recognition, Insertion and Translocation of Preproteins

The compartmentation of cells requires the accurate subcellular sorting of proteins after their synthesis on cytoplasmic ribosomes. Any cellular membrane possesses a distinct transport machinery which specifically recognizes, inserts and translocates precursor proteins destined for this particular membrane or organelle (for reviews see articles in Neupert and Lill, 1992). For instance, preproteins are inserted into the membrane of the endoplasmic reticulum and become translocated into the lumen by the action of the Sec61 and Sec63 complexes (see Rapoport et al. , this issue). For mitochondria, the transport process is particularly complex, since these organelles are bounded by two membranes, the outer and inner membrane which enclose the intermembrane space and the matrix, and separate the mitochondrion from the cytoplasm. The sub-compartmentation requires distinct sub-organellar sorting pathways which ensure the accurate distribution of the proteins. Despite the complexity of the sorting process, our knowledge about the mechanisms and the machinery of mitochondrial protein translocation has well advanced during the last decade (for recent reviews see Pfanner and Neupert, 1990; Glick and Schatz, 1991; Segui-Real et al. , 1993b; Schwarz and Neupert, 1994). In this contribution, we will focus mainly on our investigations on mechanistic aspects underlying protein transport across the mitochondrial outer membrane. The identification and properties of the components of the receptor complex have been reviewed in depth elsewhere (Kiebler et al. , 1993a; Lill et al. , 1994). For a detailed description of the events occurring at the mitochondrial inner membrane, the reader is referred to another article in this issue (Schneider et al. ).