Katsuyoshi Mihara

Structural basis of presequence recognition by the mitochondrial protein import receptor Tom20.

Most mitochondrial proteins are synthesized in the cytosol as precursor proteins with a cleavable N-terminal presequence and are imported into mitochondria. We report here the NMR structure of a general import receptor, rat Tom20, in a complex with a presequence peptide derived from rat aldehyde dehydrogenase. The cytosolic domain of Tom20 forms an all alpha-helical structure with a groove to accommodate the presequence peptide. The bound presequence forms an amphiphilic helical structure with hydrophobic leucines aligned on one side to interact with a hydrophobic patch in the Tom20 groove. Although the positive charges of the presequence are essential for import ability, presequence binding to Tom20 is mediated mainly by hydrophobic rather than ionic interactions.

Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: evidence for the 'acid chain' hypothesis

Mitochondrial precursor proteins with basic targeting signals may be transported across the outer membrane by sequential binding to acidic receptor sites of increasing affinity. To test this ‘acid chain’ hypothesis, we assayed the interaction of mitochondrial precursors with three acidic receptor domains: the cytosolic domain of Tom20 and the intermembrane space domain of Tom22 and Tim23. The apparent affinity and salt resistance of precursor binding increased in the order Tom20

A mitochondrial import factor purified from rat liver cytosol is an ATP-dependent conformational modulator for precursor proteins.

Rat liver cytosol contained an activity that stimulated the import of wheat germ lysate‐synthesized precursor proteins into mitochondria. The activity was purified 10,000‐fold from the cytosol as a homogeneous heterodimeric protein. This protein (termed mitochondrial import stimulation factor or MSF) stimulated the binding and import of mitochondrial precursor proteins. MSF was also found to recognize the presequence portion of mitochondrial precursors and catalyze the depolymerization and unfolding of in vitro synthesized mitochondrial precursor proteins in an ATP‐dependent manner; in this connection, MSF exhibited ATPase activity depending on the important‐incompetent mitochondrial precursor protein. The mitochondrial binding and import‐stimulating activities were strongly inhibited by the pretreatment of MSF with NEM, whereas the ATP‐dependent depolymerization activity was insensitive to the NEM treatment, suggesting that the process subsequent to the unfolding was inhibited with the NEM treatment. We conclude that MSF is a multifunctional cytoplasmic chaperone specific for mitochondrial protein import.

MSF, a novel cytoplasmic chaperone which functions in precursor targeting to mitochondria.

Mitochondrial import stimulation factor (MSF) unfolds wheat germ lysate synthesized aggregated mitochondrial precursor proteins and stimulates their mitochondrial import in an ATP dependent manner. Here we analysed the function of MSF mainly by utilizing chemically pure adrenodoxin precursor (pAd). MSF bound to the unfolded pAd and prevented it from losing import competence and also restored the import competence of the aggregated pAd dependent on ATP hydrolysis. The import incompetent aggregated mitochondrial precursors induced the ATPase activity of MSF and the activity was strongly inhibited by isolated mitochondrial outer membrane (OM) but not by trypsin treated outer membrane (tOM). The precursor induced ATPase activity of N‐ethylmaleimide (NEM)‐treated MSF was not inhibited by OM. In this context, the MSF‐precursor complex specifically bound to OM and binding was abolished both by the treatment of OM with trypsin and by the treatment of MSF with NEM. These results show that MSF is a novel cytoplasmic chaperone protein with a mitochondrial precursor‐targeting function.

Metaxin Is a Component of a Preprotein Import Complex in the Outer Membrane of the Mammalian Mitochondrion

Metaxin, a novel gene located between the glucocerebrosidase and thrombospondin 3 genes in the mouse, is essential for survival of the postimplantation mouse embryo. In this study, the subcellular location, domain structure, and biochemical function of metaxin were investigated. Anti-recombinant metaxin antibodies recognized 35- and 70-kDa proteins in mitochondria from various tissues; the 35-kDa protein is consistent in size with the predicted translation product of metaxin cDNA. When metaxin cDNA was transfected into COS cells, immunofluorescence staining demonstrated that the protein is located in mitochondria. Metaxin contains a putative mitochondrial outer membrane signal anchor domain at its C terminus, and a truncated form of metaxin lacking this signal anchor domain had a reduced association with mitochondria. In addition, metaxin was highly susceptible to proteases in intact mitochondria. We therefore conclude that metaxin is a mitochondrial protein that extends into the cytosol while anchored into the outer membrane at its C terminus. In its N-terminal region, metaxin shows significant sequence identity to Tom37, a component of the outer membrane portion of the mitochondrial preprotein translocation apparatus in Saccharomyces cerevisiae, but important structural differences, including apparently different mechanisms of targeting to membranes, also exist between the two proteins. Given the similar subcellular locations of metaxin and Tom37, the possible role of metaxin in mitochondrial preprotein import was investigated. Antibodies against metaxin, when preincubated with mitochondria, partially inhibited the uptake of radiolabeled preadrenodoxin into mitochondria. Metaxin is therefore the second mammalian component of the protein translocation apparatus of the mitochondrial outer membrane to be characterized at the molecular level and the first for which an inherited mutation has been described. The early embryonic lethal phenotype of mice lacking metaxin demonstrates that efficient import of proteins into mitochondria is crucial for cellular survival. The characterization of metaxin provides an opportunity to elucidate similarities and possible differences in the mechanisms of protein import between fungi and mammals and in the phenotypes of fungi and mammals lacking mitochondrial import receptors.

Binding of mitochondrial precursor proteins to the cytoplasmic domains of the import receptors Tom70 and Tom20 is determined by cytoplasmic chaperones

We have reconstituted the early steps of precursor targeting to mitochondria in a defined and soluble system consisting of the cytosolic domains of the yeast mitochondrial import receptors Tom20 and Tom70, precursor to bovine adrenal adrenodoxin (which has a cleavable targeting signal) and rat liver cytosolic chaperones hsp70 and mitochondrial import‐stimulating factor (MSF). The Tom70 domain only bound the precursor in the presence of MSF, yielding a precursor–MSF–Tom70 complex; ATP hydrolysis by MSF released MSF and generated a precursor–Tom70 complex whose formation was inhibited by an excess of a functional presequence peptide, but not by 150 mM NaCl. In the presence of the Tom20 domain, ATP caused transfer of the precursor from the precursor–MSF–Tom70 complex to Tom20. The Tom20 domain alone only bound the precursor in the presence of hsp70; hsp70 itself was not incorporated into the resulting complex. Formation of the Tom20–precursor complex was inhibited by excess presequence peptide or by 150 mM NaCl. Similar results were obtained with the ADP/ATP carrier and porin precursors, which both lack a cleaved targeting signal. Correct targeting of a precursor to mitochondrial import receptors thus requires cytosolic chaperones, irrespective of the presence or absence of a cleavable presequence.

Forced Transmembrane Orientation of Hydrophilic Polypeptide Segments in Multispanning Membrane Proteins

In a current model of integration of multispanning membrane proteins into the endoplasmic reticulum, it is proposed that the transmembrane segments show alternating translocation initiation and stop-transfer functions. Here, we present evidence for a mode of cotranslational insertion in which an internal signal-anchor sequence with Nexo/Ccyt topology confers a transmembrane disposition onto a preceding hydrophilic segment, resulting in a topology where the hydrophilic segment apparently can slip back and forth across the membrane. Our results demonstrate that hydrophobicity is not, as hitherto thought, an absolute requirement for the formation of a transmembrane segment, and suggest that integral membrane proteins may contain hydrophilic transmembrane segments with a considerable freedom to move in relation to the membrane.

Cytoplasmic chaperones determine the targeting pathway of precursor proteins to mitochondria.

Two ATP‐dependent cytosolic chaperones, mitochondrial import stimulation factor (MSF) and hsp70, are known to be involved in the import of precursor proteins into mitochondria. Hsp70 generally recognizes unfolded proteins, while MSF specifically recognizes mitochondrial precursor proteins and targets them to mitochondria in a NEM‐sensitive manner. Here we analyzed the relative contribution of these chaperones in the import process and confirmed that the precursor proteins are targeted to mitochondria via two distinct pathways: one requiring MSF and the other requiring hsp70. Both pathways depend on distinct proteinaceous components of the outer mitochondrial membrane. The MSF‐dependent pathway is NEM‐sensitive and requires the hydrolysis of extra‐mitochondrial ATP for the release of MSF from the mitochondrial import receptor, whereas the hsp70‐dependent pathway is NEM‐sensitive and does not require extra‐mitochondrial ATP. The NEM‐insensitive, hsp70‐dependent import became NEM‐sensitive depending on the amount of MSF added. The relative importance of the two pathways appears to be determined by the affinities of MSF and hsp70 for the precursor proteins.

Membrane topology of Alzheimer's disease-related presenilin 1. Evidence for the existence of a molecular species with a seven membrane-spanning and one membrane-embedded structure.

A significant member of early-onset familial type of Alzheimer’s disease cases has been shown to be caused by dominant mutations in either of the two genes encoding presenilin 1 (PS1) and presenilin 2 (PS2). These two proteins are highly homologous to each other and have been reported to be mainly localized to the membranes of intracellular compartments such as the endoplasmic reticulum. Information about the membrane topological structures of these proteins is indispensable for understanding their physiological and pathological roles. Although several models have been proposed previously, their precise membrane topologies remain unknown. In this study, we examined this issue in detail by expressing a series of C-terminally deleted PS1 mutants fused to the hydrophilic portion of Escherichia coli leader peptidase in vitro using a reticulocyte lysate in the presence of microsomal membranes. Our results predict that PS1 exists mainly in a seven membrane-spanning structure with its C-terminal end exposed to the luminal space. This was also confirmed by expressing these fusion proteins in cultured cells. We further showed that a ninth hydrophobic segment is tightly bound to the membrane without spanning it. Based on the above observations, we propose a novel “seven membrane-spanning and one membrane-embedded” topological model for presenilins.

Analysis of the Functional Domain of the Rat Liver Mitochondrial Import Receptor Tom20

Tom20 is an outer mitochondrial membrane protein and functions as a component of the import receptor complex for the cytoplasmically synthesized mitochondrial precursor proteins. It consists of the N-terminal membrane-anchor segment, the tetratricopeptide repeat (TPR) motif, a charged amino acids-rich linker segment between the membrane anchor and the TPR motif, and the C-terminal acidic amino acid cluster. To assess the functional significance of these segments in mammalian Tom20, we cloned rat Tom20 and expressed mutant rat Tom20 proteins in Δtom20 yeast cells and examined their ability to complement the defects of respiration-driven growth and mitochondrial protein import. Tom20N69, a mutant consisting of the membrane anchor and the linker segments, was targeted to mitochondria and complemented the growth and import defects as efficiently as wild-type Tom20, whereas a mutant lacking the linker segment did not. In vitro protein import into mitochondria isolated from the complemented yeast cells revealed that the precursor targeted to yeast Tom70 was efficiently imported into the mitochondria via rat Tom20N69. Thus the linker segment is essential for the function of rat Tom20, whereas the TPR motif and the C-terminal acidic amino acids are not.