Structural elements of the mitochondrial preprotein-conducting channel Tom40 dissolved by bioinformatics and mass spectrometry

Most mitochondrial proteins are imported into mitochondria from the cytosolic compartment. Proteins destined for the outer or inner membrane, the inter-membrane space, or the matrix are recognized and translocated by the TOM machinery containing the specialized protein import channel Tom40. The latter is a protein with β-barrel shape, which is suggested to have evolved from a porin-type protein. To obtain structural insights in the absence of a crystal structure the membrane topology of Tom40 from Neurospora crassa was determined by limited proteolysis combined with mass spectrometry. The results were interpreted on the basis of a structural model that has been generated for NcTom40 by using the structure of mouse VDAC-1 as a template and amino acid sequence information of approximately 270 different Tom40 and approximately 480 VDAC amino acid sequences for refinement. The model largely explains the observed accessible cleavage sites and serves as a structural basis for the investigation of physicochemical properties of the ensemble of our Tom40 sequence data set. By this means we discovered two conserved polar slides in the pore interior. One is possibly involved in the positioning of a pore-inserted helix; the other one might be important for mitochondrial pre-sequence peptide binding as it is only present in Tom40 but not in VDAC proteins. The outer surface of the Tom40 barrel reveals two conserved amino acid clusters. They may be involved in binding other components of the TOM complex or bridging components of the TIM machinery of the mitochondrial inner membrane.     

Structure of dimeric SecA, the Escherichia coli preprotein translocase motor

SecA is the preprotein translocase ATPase subunit and a superfamily 2 (SF2) RNA helicase. Here we present the 2 A crystal structures of the Escherichia coli SecA homodimer in the apo form and in complex with ATP, ADP and adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP). Each monomer contains the SF2 ATPase core (DEAD motor) built of two domains (nucleotide binding domain, NBD and intramolecular regulator of ATPase 2, IRA2), the preprotein binding domain (PBD), which is inserted in NBD and a carboxy-terminal domain (C-domain) linked to IRA2. The structures of the nucleotide complexes of SecA identify an interfacial nucleotide-binding cleft located between the two DEAD motor domains and residues critical for ATP catalysis. The dimer comprises two virtually identical protomers associating in an antiparallel fashion. Dimerization is mediated solely through extensive contacts of the DEAD motor domains leaving the C-domain facing outwards from the dimerization core. This dimerization mode explains the effect of functionally important mutations and is completely different from the dimerization models proposed for other SecA structures. The repercussion of these findings on translocase assembly and catalysis is discussed.     

Assembly of the SpoIIIE DNA translocase depends on chromosome trapping in Bacillus subtilis

Sporulation in Bacillus subtilis is an attractive system in which to study the translocation of a chromosome across a membrane. Sporulating cells contain two sister chromosomes that are condensed in an elongated axial filament with the origins of replication anchored at opposite poles of the sporangium. The subsequent formation of a septum near one pole divides the sporangium unequally into a forespore (the smaller compartment) and a mother cell. The septum forms around the filament, trapping the origin-proximal region of one chromosome in the forespore. As a consequence, the trapped chromosome transverses the septum with the remainder being left in the mother cell. Next, SpoIIIE assembles at the middle of the septum to create a translocase that pumps the origin-distal, two-thirds of the chromosome into the forespore. Here, we address the question of how the DNA translocase assembles and how it localizes to the septal midpoint. We present evidence that DNA transversing the septum is an anchor that nucleates the formation of the DNA translocase. We propose that DNA anchoring is responsible for the assembly of other SpoIIIE-like DNA translocases, such as those that remove trapped chromosomes from the division septum of cells undergoing binary fission.     

Assembly of the three small Tim proteins precedes docking to the mitochondrial carrier translocase

The mitochondrial intermembrane space contains a family of small Tim proteins that function as essential chaperones for protein import. The soluble Tim9-Tim10 complex transfers hydrophobic precursor proteins through the aqueous intermembrane space to the carrier translocase of the inner membrane (TIM22 complex). Tim12, a peripheral membrane subunit of the TIM22 complex, is thought to recruit a portion of Tim9-Tim10 to the inner membrane. It is not known, however, how Tim12 is assembled. We have identified a new intermediate in the biogenesis pathway of Tim12. A soluble form of Tim12 first assembles with Tim9 and Tim10 to form a Tim12-core complex. Tim12-core then docks onto the membrane-integrated subunits of the TIM22 complex to form the holo-translocase. Thus, the function of Tim12 in linking soluble and membrane-integrated subunits of the import machinery involves a sequential assembly mechanism of the translocase through a soluble intermediate complex of the three essential small Tim proteins.     

Multiple variants of the human presequence translocase motor subunit Magmas govern the mitochondrial import

Mitochondrial protein translocation is an intricately regulated process that requires dedicated translocases at the outer and inner membranes. The presequence translocase complex, translocase of the inner membrane 23, facilitates most of the import of preproteins containing presequences into the mitochondria, and its primary structural organization is highly conserved. As part of the translocase motor, two J-proteins, DnaJC15 and DnaJC19, are recruited to form two independent translocation machineries (translocase A and translocase B, respectively). On the other hand, the J-like protein subunit of translocase of the inner membrane 23, Mitochondria-associated granulocyte-macrophage colony-stimulating factor signaling molecule (Magmas) (orthologous to the yeast subunit Pam16), can regulate human import-motor activity by forming a heterodimer with DnaJC19 and DnaJC15. However, the precise coordinated regulation of two human import motors by a single Magmas protein is poorly understood. Here, we report two additional Magmas variants (Magmas-1 and Magmas-2) constitutively expressed in the mammalian system. Both the Magmas variants are functional orthologs of Pam16 with an evolutionarily conserved J-like domain critical for cell survival. Moreover, the Magmas variants are peripherally associated with the inner membrane as part of the human import motor for translocation. Our results demonstrate that Magmas-1 is predominantly recruited to translocase B, whereas Magmas-2 is majorly associated with translocase A. Strikingly, both the variants exhibit differential J-protein inhibitory activity in modulating import motor, thereby regulating overall translocase function. Based on our findings, we hypothesize that additional Magmas variants are of evolutionary significance in humans to maximize protein import in familial-linked pathological conditions.     

Assembly of DNA onto the histone octamer facilitates the B-to-Z transition

Nucleosomal core particles containing the right- and left-handed conformations of DNA were examined for their ability to support the BZ or ZB transition. Nucleosomes were assembled onto the B- and Z-conformations of poly[d(Gm5C)] and the B-conformation of poly[d(GC)] as previously described (1). Absorbance and circular dichroic spectroscopy indicated that the DNA on all three core particle populations could undergo the conformational BZ transition. Further, the right- to left-handed transition for both poly[d(Gm5C)] and poly[d(GC)] appeared to be facilitated by the DNAs association with the histone octamer. The DNA remained associated with the protein core subsequent to the transition, and electron microscopy and sedimentation velocity analysis indicated that there were no gross changes in nucleosomal structure. However, a change in the sedimentation value of the poly[d(Gm5C)] core particles was detected when the conformation of the DNA was altered from B to Z, resulting in a lower S20,w value for the Z-form particles than for the corresponding B-form particles.     

Structural basis for signal-sequence recognition by the translocase motor SecA as determined by NMR

Recognition of signal sequences by cognate receptors controls the entry of virtually all proteins to export pathways. Despite its importance, this process remains poorly understood. Here, we present the solution structure of a signal peptide bound to SecA, the 204 kDa ATPase motor of the Sec translocase. Upon encounter, the signal peptide forms an alpha-helix that inserts into a flexible and elongated groove in SecA. The mode of binding is bimodal, with both hydrophobic and electrostatic interactions mediating recognition. The same groove is used by SecA to recognize a diverse set of signal sequences. Impairment of the signal-peptide binding to SecA results in significant translocation defects. The C-terminal tail of SecA occludes the groove and inhibits signal-peptide binding, but autoinhibition is relieved by the SecB chaperone. Finally, it is shown that SecA interconverts between two conformations in solution, suggesting a simple mechanism for polypeptide translocation.     

A presequence- and voltage-sensitive channel of the mitochondrial preprotein translocase formed by Tim23.

Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes. The channel across the inner membrane is formed by the presequence translocase, which consists of roughly six distinct subunits; however, it is not known which subunits actually form the channel. Here we report that purified Tim23 forms a hydrophilic, approximately 13-24 A wide channel characteristic of the mitochondrial presequence translocase. The Tim23 channel is cation selective and activated by a membrane potential and presequences. The channel is formed by the C-terminal domain of Tim23 alone, whereas the N-terminal domain is required for selectivity and a high-affinity presequence interaction. Thus, Tim23 forms a voltage-sensitive high-conductance channel with specificity for mitochondrial presequences.     

Sorting switch of mitochondrial presequence translocase involves coupling of motor module to respiratory chain

The mitochondrial presequence translocase transports preproteins to either matrix or inner membrane. Two different translocase forms have been identified: the matrix transport form, which binds the heat-shock protein 70 (Hsp70) motor, and the inner membrane–sorting form, which lacks the motor but contains translocase of inner mitochondrial membrane 21 (Tim21). The sorting form interacts with the respiratory chain in a Tim21-dependent manner. It is unknown whether the respiratory chain–bound translocase transports preproteins and how the switch between sorting form and motor form occurs. We report that the respiratory chain–bound translocase contains preproteins in transit and, surprisingly, not only sorted but also matrix-targeted preproteins. Presequence translocase-associated motor (Pam) 16 and 18, two regulatory components of the six-subunit motor, interact with the respiratory chain independently of Tim21. Thus, the respiratory chain–bound presequence translocase is not only active in preprotein sorting to the inner membrane but also in an early stage of matrix translocation. The motor does not assemble en bloc with the translocase but apparently in a step-wise manner with the Pam16/18 module before the Hsp70 core.     

Tim14, a novel key component of the import motor of the TIM23 protein translocase of mitochondria

The TIM23 translocase mediates the deltaPsi- and ATP-dependent import of proteins into mitochondria. We identified Tim14 as a novel component of the TIM23 translocase. Tim14 is an integral protein of the inner membrane with a typical J-domain exposed to the matrix space. TIM14 genes are present in the genomes of virtually all eukaryotes. In yeast, Tim14 is essential for viability. Mitochondria from cells depleted of Tim14 are deficient in the import of proteins mediated by the TIM23 complex. In particular, import of proteins that require the action of mtHsp70 is affected. Tim14 interacts with Tim44 and mtHsp70 in an ATP-dependent manner. A mutation in the HPD motif of the J-domain of Tim14 is lethal. Thus, Tim14 is a constituent of the mitochondrial import motor. We propose a model in which Tim14 is required for the activation of mtHsp70 and enables this chaperone to act in a rapid and regulated manner in the Tim44-mediated trapping of unfolded preproteins entering the matrix.     

Differential channeling of sensory stimuli onto a motor neuron in the leech

We studied a specific sensory-motor pathway in the isolated leech ganglia. Pressure-sensitive mechanosensory neurons were stimulated with trains of action potentials at 5–20 Hz while recording the responses of the annulus erector motorneurons that control annuli erection. The response of the annulus erector neurons was a succession of excitatory postsynaptic potentials followed by inhibitory postsynaptic potentials. The excitatory postsynaptic potentials had a brief time-course while the inhibitory postsynaptic potentials had a prolonged time-course that enabled their temporal summation. Thus, the net effect of pressure-sensitive neuron stimulation on the annulus erector neurons was inhibitory. Both phases of the response were mediated by chemical transmission; the excitatory postsynaptic potentials were transmitted via a monosynaptic pathway, and the inhibitory postsynaptic potentials via a polysynaptic one. The pattern of expression of this dual response depended on the field of innervation of the sensory neuron and it was under the influence of cell 151, a non-spiking interneuron, that could regulate the expression of the hyperpolarization. The interaction between pressure-sensitive neurons and annulus erector neuron reveals how sensory specificity, connectivity pattern and regulatory elements interplay in a specific sensory-motor network. Accepted: 6 November 1998     

Assembly and stability of flagellar motor in Escherichia coli

Bacterial flagellar motor is a highly ordered and complex supramolecular structure that powers rotation of flagella and serves as a type III export apparatus for flagellar assembly. Motor biogenesis represents a formidable example of self-assembly, but little is known about early steps of the motor structure formation. Here we used a combination of fluorescence microscopy techniques to dissect the order of the motor assembly in Escherichia coli cells, to map in vivo the underlying protein interactions and to investigate dynamics of protein exchange in the assembled motor structure. Our data suggest that motor self-assembly is initiated by oligomerization of the membrane export apparatus protein FlhA, which is followed by the recruitment of the MS ring component FliF and by the ordered association of other motor proteins. The assembly process combines the hierarchy with cooperativity, whereby the association of each subsequent motor structure stabilizes the growing assembly. Our results provide a novel and so far the most complete view of the early steps in flagellar motor assembly and improve understanding of the motor structure and regulation.     

The mitochondrial presequence translocase: an essential role of Tim50 in directing preproteins to the import channel

Mitochondrial proteins with N-terminal targeting signals are transported across the inner membrane via the presequence translocase, which consists of membrane-integrated channel proteins and the matrix Hsp70 import motor. It has not been known how preproteins are directed to the import channel. We have identified the essential protein Tim50, which exposes its major domain to the intermembrane space. Tim50 interacts with preproteins in transit and directs them to the channel protein Tim23. Inactivation of Tim50 strongly inhibits the import of preproteins with a classical matrix-targeting signal, while preproteins carrying an additional inner membrane-sorting signal do not strictly depend on Tim50. Thus, Tim50 is crucial for guiding the precursors of matrix proteins to their insertion site in the inner membrane.     

Spatial distribution of synapses onto thoracic motor neurones in locusts

Summary Aggregates of synaptic vesicles, stained black by the zinc iodide-osmium procedure, can be visualised with the light microscope in 1 m plastic sections. This allows the main branches of a neurone to be reconstructed relatively rapidly and the associated vesicle aggregates to be plotted. By resectioning, the identity of the vesicle aggregates has been confirmed with the electron microscope. Two flight motor neurones in the mesothoracic ganglion of the locust have been examined. One is identified as a dorsal longitudinal muscle motor neurone (muscle 112) and the other is probably a subalar neurone (muscle 99). Both have a large density of vesicle aggregates on the neuropilar segment, the widest part of the main neuronal axis, but few on the neurite within 250 m of the cell body. The larger branches arising from the neuropilar segment tend to have a lower density of aggregates than fine branches, which suggests that synapses to the branches may occur mainly on the distal twigs. These results are an important preliminary step in determining the integrative functions of such neurones and have immediate implications in the interpretation of microelectrode recordings.JSA is supported by grant KU 240/3 from the Deutsche Forschungsgemeinschaft to Dr. W. Kutsch. We thank Mrs. Christine Davies for valuable assistance with the resectioning technique.     

Assembly organization of the motor region of the cerebral cortex in adult humans

Field IV and adjacent areas of fields VI, VI op, VI p of the human brain cortex were examined. Frontal, sagittal, tangential sections stained after the techniques of Nissle, Brachet, Klüver--Barer, Cajal and Peters demonstrated the motor cortex to contain a great number of local, with rare overlapping, groups of cellular columns, each of them being surrounded with a peculiar vessel-fibrillar capsule. A morphological unit resulted from topographic interdependens of neurons, vessels and fibres is considered as a neuronal assembly which occupies in the cortical space a figure of truncated upset cone or cylinder. Neuronal assemblies are distributed along radiate fibres and radial vessels, do not form regularly alternated raws, central axis of every assembly is perpendicular to the tangent line drawn in the given point of the cortical surface.     

Assembly of motor proteins, PomA and PomB, in the Na+-driven stator of the flagellar motor

PomA and PomB are transmembrane proteins that form the stator complex in the sodium-driven flagellar motor of Vibrio alginolyticus and are believed to surround the rotor part of the flagellar motor. We constructed and observed green fluorescent protein (GFP) fusions of the stator proteins PomA and PomB in living cells to clarify how stator proteins are assembled and installed into the flagellar motor. We were able to demonstrate that GFP-PomA and GFP-PomB localized to a cell pole dependent on the presence of the polar flagellum. Localization of the GFP-fused stator proteins required their partner subunit, PomA or PomB, and the C-terminal domain of PomB, which has a peptidoglycan-binding motif. Each of the GFP-fused stator proteins was co-isolated with its partner subunit from detergent-solubilized membrane. From these lines of evidence, we have demonstrated that the stator proteins are incorporated into the flagellar motor as a PomA/PomB complex and are fixed to the cell wall via the C-terminal domain of PomB.     

MotX, the channel component of the sodium-type flagellar motor.

Thrust for propulsion of flagellated bacteria is generated by rotation of a propeller, the flagellum. The power to drive the polar flagellar rotary motor of Vibrio parahaemolyticus is derived from the transmembrane potential of sodium ions. Force is generated by the motor on coupling of the movement of ions across the membrane to rotation of the flagellum. A gene, motX, encoding one component of the torque generator has been cloned and sequenced. The deduced protein sequence is 212 amino acids in length. MotX was localized to the membrane and shown to interact with MotY, which is the presumed stationary component of the motor. Overproduction of MotX, but not that of a nonfunctional mutant MotX, was lethal to Escherichia coli. The rate of lysis caused by induction of motX was proportional to the sodium ion concentration. Li+ and K+ substituted for Na+ to promote lysis, while Ca2+ did not enhance lysis. Protection from the lethal effects of induction of motX was afforded by the sodium channel blocker amiloride. The data suggest that MotX forms a sodium channel. The deduced protein sequence for MotX shows no homology to its ion-conducting counterpart in the proton-driven motor; however, in possessing only one hydrophobic domain, it resembles other channels formed by small proteins with single membrane-spanning domains.     

Assembly of presynaptic filaments. Factors affecting the assembly of RecA protein onto single-stranded DNA

We have previously shown that the assembly of RecA protein onto single-stranded DNA (ssDNA) facilitated by SSB protein occurs in three steps: (1) rapid binding of SSB protein to the ssDNA; (2) nucleation of RecA protein onto this template; and (3) co-operative polymerization of additional RecA protein to yield presynaptic filaments. Here, electron microscopy has been used to further explore the parameters of this assembly process. The optimal extent of presynaptic filament formation required at least one RecA protein monomer per three nucleotides, high concentrations of ATP (greater than 3 mM in the presence of 12 mM-Mg2+), and relatively low concentrations of SSB protein (1 monomer per 18 nucleotides). Assembly was depressed threefold when SSB protein was added to one monomer per nine nucleotides. These effects appeared to be exerted at the nucleation step. Following nucleation, RecA protein assembled onto ssDNA at net rates that varied from 250 to 900 RecA protein monomers per minute, with the rate inversely related to the concentration of SSB protein. Combined sucrose sedimentation and electron microscope analysis established that SSB protein was displaced from the ssDNA during RecA protein assembly.