Biogenesis of the mitochondrial TOM complex

The translocase at the outer membrane of mitochondria (TOM complex) mediates the initial steps of the import of preproteins into the organelle, which are essential for mitochondrial biogenesis and, therefore, for eukaryotic cell viability. The TOM complex is a multisubunit molecular machine with a dynamic structure. The biogenesis of TOM is of special interest because the complex is required for its own biogenesis. This article describes the mechanisms by which Tom components are targeted to the mitochondria and inserted into the outer membrane. The assembly of newly synthesized subunits into the functional TOM complex might occur via assembly intermediates that are in equilibrium with the mature complex.     

Biogenesis of the Semliki Forest virus RNA replication complex

The nonstructural (ns) proteins nsP1 to -4, the components of Semliki Forest virus (SFV) RNA polymerase, were localized in infected cells by confocal microscopy using double labeling with specific antisera against the individual ns proteins. All ns proteins were associated with large cytoplasmic vacuoles (CPV), the inner surfaces of which were covered by small invaginations, or spherules, typical of alphavirus infection. All ns proteins were localized by immuno-electron microscopy (EM) to the limiting membranes of CPV and to the spherules, together with newly labeled viral RNA. Along with earlier observations by EM-autoradiography (P. M. Grimley, I. K. Berezesky, and R. M. Friedman, J. Virol. 2:326–338, 1968), these results suggest that individual spherules represent template-associated RNA polymerase complexes. Immunoprecipitation of radiolabeled ns proteins showed that each antiserum precipitated the other three ns proteins, implying that they functioned as a complex. Double labeling with organelle-specific and anti-ns-protein antisera showed that CPV were derivatives of late endosomes and lysosomes. Indeed, CPV frequently contained endocytosed bovine serum albumin-coated gold particles, introduced into the medium at different times after infection. With time, increasing numbers of spherules were also observed on the cell surfaces; they were occasionally released into the medium, probably by secretory lysosomes. We suggest that the spherules arise by primary assembly of the RNA replication complexes at the plasma membrane, guided there by nsP1, which has affinity to lipids specific for the cytoplasmic leaflet of the plasma membrane. Endosomal recycling and fusion of CPV with the plasma membrane can circulate spherules between the plasma membrane and the endosomal-lysosomal compartment.     

Biogenesis of Tom40, core component of the TOM complex of mitochondria.

Tom40 is an essential component of the preprotein translocase of the mitochondrial outer membrane (TOM complex) in which it constitutes the core element of the protein conducting pore. We have investigated the biogenesis of Tom40. Tom40 is inserted into the outer membrane by the TOM complex. Initially, Tom40 is bound as a monomer at the mitochondrial surface. The import receptor Tom20 is involved in this initial step; it stimulates both binding and efficient insertion of the Tom40 precursor. This step is followed by the formation of a further intermediate at which the Tom40 precursor is partially inserted into the outer membrane. Finally, Tom40 is integrated into preexisting TOM complexes. Efficient import appears to require the Tom40 precursor to be in a partially folded conformation. Neither the NH(2) nor the COOH termini are necessary to target Tom40 to the outer membrane. However, the NH(2)-terminal segment is required for Tom40 to become assembled into the TOM complex. A model for the biogenesis of Tom40 is presented.     

Biogenesis of a photosystem I light-harvesting complex. Evidence for a membrane intermediate.

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chlorophyll complexes produced by detergent solubilization of PSI. Two types of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane intermediate. First, when CAB-7p is briefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PSI does not require stroma or ATP. Second, trypsin treatment of thylakoids containing radiolabeled CAB-7p indicates that there are at least two membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this to mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observation that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7-kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/BgI2p, that was able to integrate into thylakoid membranes but was unable to assemble into PSI. The membrane form of CAB-7/BgI2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We suggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing gels and its susceptibility to protease cleavage, we suggest that the intermediate form is LHCI-680. We propose the following distinct stages in the biogenesis of LHCI: (a) apoprotein is integrated into the thylakoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680, and (c) LHCI-680 assembles into the native PSI complex.     

Biogenesis of the cytochrome bc1 complex in isolated rat hepatocytes

Isolated rat hepatocytes were labeled with [³⁵S]methionine in the absence or presence of cycloheximide or chloramphenicol. The cytochrome $\text{bc}$₁ complex was isolated from labeled cells by a micromethod and analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. All subunits except the two smallest, subunits VII and VIII, were labeled in the absence of translational inhibitors. In the presence of cycloheximide only subunit III (molecular weight, 30 000) was labeled. This polypeptide, identified as an apo-cytochrome b, was weakly labeled with [³⁵S]methionine in the presence of cycloheximide, indicating a strict dependence of cytoplasmically synthesized products for its assembly. In the presence of chloramphenicol, labeling was inhibited only in subunit III.     

Reprint of: Biogenesis of the cytochrome bc1 complex and role of assembly factors

The cytochrome bc₁ complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc₁ complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc₁ complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc₁ complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc₁ complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron–sulfur protein and its role in completing the assembly of functional bc₁ complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Biogenesis of the bacterial pilus.

The assembly of surface structures in gram-negative bacteria requires specialized secretion and chaperone systems localized on both sides of the cytoplasmic membrane. Major advances have been made over the last year in understanding how these systems form part of a general strategy used by bacteria to cap and target interactive subunits imported into the periplasmic space to outer membrane uncapping and assembly sites.     

Biogenesis of ER-to-Golgi transport carriers: complex roles of COPII in ER export

It is widely believed that membrane traffic occurs by vesicular transport between successive compartments of the secretory pathway. Coat complexes function to collect cargo from donor membranes and deform them to generate transport vesicles with a diameter of 60-80 nm. Recent data argue in favour of a new model for export of secretory cargo from the endoplasmic reticulum, in which tubular extensions are protruded and subsequently matured into independent ER-to-Golgi transport carriers. Here, we examine the evidence for this controversial hypothesis.     

Ultrastructural basis of the coordination of ciliary movement. Contractile function of the interconnecting cross-striated roots in ependymal epithelium

A hypothesis for contraction was suggested (molecular basis of the transmission of ciliary coordination) by means of the slipping and interdigitation of the microfilaments of one period with those of the adjacent periods, occurring at the level of the periodic band. The amplitude of the period is greater in the relaxed than in the contracted state, while the reverse is true with the periodic band amplitude. The microfilament length is constant, probably varying with the species. This hypothesis of the slipping and interdigitation of the microfilaments would be the basis of the ciliary coordination from cilium to cilium and from cell to cell, because of the anchoring of the bunches in the peribasal complexes and in the adherent connection complexes, respectively.     

Biogenesis of the cytochrome bc1 complex and role of assembly factors

The cytochrome bc₁ complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc₁ complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc₁ complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc₁ complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc₁ complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron–sulfur protein and its role in completing the assembly of functional bc₁ complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

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.     

Reprint of: Biogenesis of the cytochrome bc(1) complex and role of assembly factors

The cytochrome bc(1) complex is an essential component of the electron transport chain in most prokaryotes and in eukaryotic mitochondria. The catalytic subunits of the complex that are responsible for its redox functions are largely conserved across kingdoms. In eukarya, the bc(1) complex contains supernumerary subunits in addition to the catalytic core, and the biogenesis of the functional bc(1) complex occurs as a modular assembly pathway. Individual steps of this biogenesis have been recently investigated and are discussed in this review with an emphasis on the assembly of the bc(1) complex in the model eukaryote Saccharomyces cerevisiae. Additionally, a number of assembly factors have been recently identified. Their roles in bc(1) complex biogenesis are described, with special emphasis on the maturation and topogenesis of the yeast Rieske iron-sulfur protein and its role in completing the assembly of functional bc(1) complex. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Biogenesis of mitochondria. 30

Summary Mitchondrial gene recombination in S. cerevisiae was investigated using four combinations of mitochondrial markers: [oli1-r ery1-r], [oli1-r spi2-r], [oli1-r spi3-r] and [oli1-r spi4-r] in cis bifactorial crosses to [oli-s ery-s spi-s] strains. A number of sensitive strains including representatives of both mating types and of diverse origin were used. The crosses were analysed for frequency and polarity of mitochondrial gene recombination as well as the frequency of transmission into the diploid progeny of individual mitochondrial determinants.The results show that the polarity of recombination varied markedly in crosses between a single pair of mitochondrial markers and many unrelated sensitive strains. For example, one series of crosses included polarity values of 1.7,0.34,0.081, and 0.021. Furthermore, there was also considerable variability in frequency of recombination and frequency of transmission of individual markers and these frequencies were not correlated in many cases with polarity values. However, in certain other crosses involving different marker combinations there was a correlation between extreme polarity, high recombination frequency and high transmission frequency of one marker. The results are not compatible with polarity being determined by a simple mitochondrial sex factor and suggest that several different interactions are operating which might include nuclear phenomena.