TbLOK1/ATOM19 is a novel subunit of the noncanonical mitochondrial outer membrane protein translocase of Trypanosoma brucei

TbLOK1 has previously been characterized as a trypanosomatid‐specific mitochondrial outer membrane protein whose ablation caused a collapse of the mitochondrial network, disruption of the membrane potential and loss of mitochondrial DNA. Here we show that ablation of TbLOK1 primarily abolishes mitochondrial protein import, both in vivo and in vitro. Co‐immunprecipitations together with blue native gel analysis demonstrate that TbLOK1 is a stable and stoichiometric component of the archaic protein translocase of the outer membrane (ATOM), the highly diverged functional analogue of the TOM complex in other organisms. Furthermore, we show that TbLOK1 together with the other ATOM subunits forms a complex functional network where ablation of individual subunits either causes degradation of a specific set of other subunits or their exclusion from the ATOM complex. In summary these results establish that TbLOK1 is an essential novel subunit of the ATOM complex and thus that its primary molecular function is linked to mitochondrial protein import across the outer membrane. The previously described phenotypes can all be explained as consequences of the lack of mitochondrial protein import. We therefore suggest that in line with the nomenclature of the ATOM complex subunits, TbLOK1 should be renamed to ATOM19.     

The fine structure of the fertilization membrane of the feather star Comanthus japonica (Echinodermata: Crinoidea)

Scanning electron microscopy reveals that the outer surface of the Comanthus fertilization membrane bears a network of 14 µ high ridges outlining rows of polygonal facets; however, no spines are present. The so-called spines reported previously by light microscopists were simply optical cross sections of the ridges. Transmission electron microscopy reveals that the fertilization membrane has (1) an outer component consisting mainly of dense, granular material, and (2) an inner component consisting mainly of an interlacing network of 50 Å fibers of moderate electron density. Associated with the fibers of the inner component are dense granular rods and dense lentoid discs.     

Vascularized outer-table calvarial bone flaps

Based on an anatomic study of the vascularization of the calvarium in cadavers, a technique for the transfer of vascularized outer-table calvarial bone has been developed. The outer table of the calvarium receives numerous small perforators from its overlying periosteum. The periosteum is continuous with a distinct fascial layer overlying the temporal aponeurosis which we have termed the innominate fascia. Because of a network of anastomosing vessels from proximal branches of the superficial temporal artery and perforating branches of the deep temporal artery, the outer table of the calvarium can be carried on a pedicle which contains the temporal aponeurosis, innominate fascia, and periosteum. Thirty-seven vascularized outer-table calvarial bone flaps have been performed for a variety of craniofacial reconstructive deformities. Remarkable stability and lack of resorption have led the authors to favor this method of reconstruction particularly in poorly vascularized or previously infected recipient beds.     

Identification of intermediates in the pathway of protein import into chloroplasts and their localization to envelope contact sites

We have used a hybrid precursor protein to study the pathway of protein import into chloroplasts. This hybrid (pS/protA) consists of the precursor to the small subunit of Rubisco (pS) fused to the IgG binding domains of staphylococcal protein A. The pS/protA is efficiently imported into isolated chloroplasts and is processed to its mature form (S/protA). In addition to the mature stromal form, two intermediates in the pathway of pS/protA import were identified at early time points in the import reaction. The first intermediate represents unprocessed pS/protA bound to the outer surface of the chloroplast envelope and is analogous to a previously characterized form of pS that is specifically bound to the chloroplast surface and can be subsequently translocated in the stroma (Cline, K., M. Werner-Washburne, T. H. Lubben, and K. Keegstra. 1985. J. Biol. Chem. 260:3691-3696.) The second intermediate represents a partially translocated form of the precursor that remains associated with the envelope membrane. This form is processed to mature S/protA, but remains susceptible to exogenously added protease in intact chloroplasts. We conclude that the envelope associated S/protA is spanning both the outer and inner chloroplast membranes en route to the stroma. Biochemical and immunochemical localization of the two translocation intermediates indicates that both forms are exposed at the surface of the outer membrane at sites where the outer and inner membrane are closely apposed. These contact zones appear to be organized in a reticular network on the outer envelope. We propose a model for protein import into chloroplasts that has as its central features two distinct protein conducting channels in the outer and inner envelope membranes, each gated open by a distinct subdomain of the pS signal sequence.     

Direct binding of Cenp-C to the Mis12 complex joins the inner and outer kinetochore

Kinetochores are proteinaceous scaffolds implicated in the formation of load-bearing attachments of chromosomes to microtubules during mitosis. Kinetochores contain distinct chromatin- and microtubule-binding interfaces, generally defined as the inner and outer kinetochore, respectively (reviewed in). The constitutive centromere-associated network (CCAN) and the Knl1-Mis12-Ndc80 complexes (KMN) network are the main multisubunit protein assemblies in the inner and outer kinetochore, respectively. The point of contact between the CCAN and the KMN network is unknown. Cenp-C is a conserved CCAN component whose central and C-terminal regions have been implicated in chromatin binding and dimerization. Here, we show that a conserved motif in the N-terminal region of Cenp-C binds directly and with high affinity to the Mis12 complex. Expression in HeLa cells of the isolated N-terminal motif of Cenp-C prevents outer kinetochore assembly, causing chromosome missegregation. The KMN network is also responsible for kinetochore recruitment of the components of the spindle assembly checkpoint, and we observe checkpoint impairment in cells expressing the Cenp-C N-terminal segment. Our studies unveil a crucial and likely universal link between the inner and outer kinetochore.     

Outer membrane proteins of thebacteroides fragilis group

The Triton-insoluble outer membrane proteins of members of the obligately anaerobicBacteroides fragilis group were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The application of previously published methods for the preparation of outer membranes from these anaerobes led to relatively low recoveries of outer membrane material. Additionally, the outer membrane recovered was not qualitatively or quantitatively representative of the total outer membrane. A method previously devised for the isolation of the outer membrane proteins fromEscherichia coli was used. By this method, the members of theB. fragilis group were found to possess a striking capacity to alter their cell-surface proteins in response to cultivation in different media.     

Architecture of the cell envelope of Chlamydia psittaci 6BC.

The cysteine-rich envelope proteins of the elementary body form of chlamydiae are thought to be located in the outer membrane on the basis of their insolubility in the weak anionic detergent N-lauryl sarcosinate (Sarkosyl). We found, however, that the insolubility of the small (EnvA) and the large (EnvB) cysteine-rich proteins of Chlamydia psittaci 6BC in Sarkosyl is dependent on the maintenance of a supramolecular disulfide-cross-linked complex and is unlikely to be a valid indicator of outer membrane location. Consequently, we used other methods to characterize the architecture of the cell envelope of C. psittaci 6BC. We found that disulfide-reduced EnvA, previously shown to be a lipoprotein, segregated into the detergent phase during Triton X-114 partitioning experiments and was recovered from the membrane fraction of elementary bodies lysed by nondetergent means. In contrast, disulfide-reduced EnvB segregated to the aqueous phase in partitioning experiments and was found in the soluble fraction of elementary bodies lysed in the absence of detergents. The hydrophobic affinity probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine labeled the major outer membrane protein and EnvA but did not label EnvB. Treatment of intact elementary bodies of C. psittaci with trypsin had no effect on the cysteine-rich proteins, although the major outer membrane protein was partially degraded. On the basis of these and other observations, we propose that EnvA is anchored to the outer membrane by its lipid moiety, with a hydrophilic peptide portion extending into the periplasm, and that EnvB is located exclusively within the periplasm. We further propose that disulfide-cross-linked polymers of EnvB are the functional equivalent of peptidoglycan, forming a disulfide-cross-linked network with the periplasmic domains of EnvA and other membrane proteins, which accounts for the osmotic stability of elementary bodies.     

The cell surface expression of group 2 capsular polysaccharides in Escherichia coli: the role of KpsD, RhsA and a multi-protein complex at the pole of the cell

The export of large negatively charged capsular polysaccharides across the outer membrane represents a significant challenge to Gram negative bacteria. In the case of Escherichia coli group 2 capsular polysaccharides, the mechanism of export across the outer membrane was unknown, with no identified candidate outer membrane proteins. In this paper we demonstrate that the KpsD protein, previously believed to be a periplasmic protein, is an outer membrane protein involved in the export of group 2 capsular polysaccharides across the outer membrane. We demonstrate that KpsD and KpsE are located at the poles of the cell and that polysaccharide biosynthesis and export occurs at these polar sites. By in vivo chemical cross-linking and MALDI-TOF-MS analysis we demonstrate the presence of a multi-protein biosynthetic/export complex in which cytoplasmic proteins involved in polysaccharide biosynthesis could be cross-linked to proteins involved in export across the inner and outer membranes. In addition, we show that the RhsA protein, of previously unknown function, could be cross-linked to the complex and that a rhsA mutation reduces K5 biosynthesis suggesting a role for RhsA in coupling biosynthesis and export.     

A Highly Redundant Gene Network Controls Assembly of the Outer Spore Wall in S. cerevisiae

The spore wall of Saccharomyces cerevisiae is a multilaminar extracellular structure that is formed de novo in the course of sporulation. The outer layers of the spore wall provide spores with resistance to a wide variety of environmental stresses. The major components of the outer spore wall are the polysaccharide chitosan and a polymer formed from the di-amino acid dityrosine. Though the synthesis and export pathways for dityrosine have been described, genes directly involved in dityrosine polymerization and incorporation into the spore wall have not been identified. A synthetic gene array approach to identify new genes involved in outer spore wall synthesis revealed an interconnected network influencing dityrosine assembly. This network is highly redundant both for genes of different activities that compensate for the loss of each other and for related genes of overlapping activity. Several of the genes in this network have paralogs in the yeast genome and deletion of entire paralog sets is sufficient to severely reduce dityrosine fluorescence. Solid-state NMR analysis of partially purified outer spore walls identifies a novel component in spore walls from wild type that is absent in some of the paralog set mutants. Localization of gene products identified in the screen reveals an unexpected role for lipid droplets in outer spore wall formation.     

ODA16p, a Chlamydomonas flagellar protein needed for dynein assembly

Dynein motors of cilia and flagella function in the context of the axoneme, a very large network of microtubules and associated proteins. To understand how dyneins assemble and attach to this network, we characterized two Chlamydomonas outer arm dynein assembly (oda) mutants at a new locus, ODA16. Both oda16 mutants display a reduced beat frequency and altered swimming behavior, similar to previously characterized oda mutants, but only a partial loss of axonemal dyneins as shown by both electron microscopy and immunoblots. Motility studies suggest that the remaining outer arm dyneins on oda16 axonemes are functional. The ODA16 locus encodes a 49-kDa WD-repeat domain protein. Homologues were found in mammalian and fly databases, but not in yeast or nematode databases, implying that this protein is only needed in organisms with motile cilia or flagella. The Chlamydomonas ODA16 protein shares 62% identity with its human homologue. Western blot analysis localizes more than 90% of ODA16p to the flagellar matrix. Because wild-type axonemes retain little ODA16p but can be reactivated to a normal beat in vitro, we hypothesize that ODA16p is not an essential dynein subunit, but a protein necessary for dynein transport into the flagellar compartment or assembly onto the axoneme.     

Chlamydomonas flagellar outer row dynein assembly protein ODA7 interacts with both outer row and I1 inner row dyneins

We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.     

Proteomics of photoreceptor outer segments identifies a subset of SNARE and Rab proteins implicated in membrane vesicle trafficking and fusion

The outer segment is a specialized compartment of vertebrate rod and cone photoreceptor cells where phototransduction takes place. In rod cells it consists of an organized stack of disks enclosed by a separate plasma membrane. Although most proteins involved in phototransduction have been identified and characterized, little is known about the proteins that are responsible for outer segment structure and renewal. In this study we used a tandem mass spectrometry-based proteomics approach to identify proteins in rod outer segment preparations as an initial step in defining their roles in photoreceptor structure, function, renewal, and degeneration. Five hundred and sixteen proteins were identified including 41 proteins that function in rod and cone phototransduction and the visual cycle and most proteins previously shown to be involved in outer segment structure and metabolic pathways. In addition, numerous proteins were detected that have not been previously reported to be present in outer segments including a subset of Rab and SNARE proteins implicated in vesicle trafficking and membrane fusion. Western blotting and immunofluorescence microscopy confirmed the presence of Rab 11b, Rab 18, Rab 1b, and Rab GDP dissociation inhibitor in outer segments. The SNARE proteins, VAMP2/3, syntaxin 3, N-ethylmaleimide-sensitive factor, and Munc 18 detected in outer segment preparations by mass spectrometry and Western blotting were also observed in outer segments by immunofluorescence microscopy. Syntaxin 3 and N-ethylmaleimide- sensitive factor had a restricted localization at the base of the outer segments, whereas VAMP2/3 and Munc 18 were distributed throughout the outer segments. These results suggest that Rab and SNARE proteins play a role in vesicle trafficking and membrane fusion as part of the outer segment renewal process. The data set generated in this study is a valuable resource for further analysis of photoreceptor outer segment structure and function.     

Whole-Depth Change in Bovine Zona Pellucida Biomechanics after Fertilization: How Relevant in Hindering Polyspermy?

Polyspermy is a common problem in bovine in vitro fertilization (IVF) and has a still unclear etiology. In this specie, after IVF, despite the lack of a biochemical post-fertilization hardening, the stiffness of the outer ZP layer is significantly increased. Therefore, polyspermy might be related to an incomplete or insufficient stiffening of the ZP. We obtained, by using atomic force spectroscopy in physiological conditions, a complete characterization of the biomechanical changes of the inner and outer ZP layers occurring during oocyte maturation/fertilization and correlated them to the ultrastructural changes observed by transmission electron microscopy using ruthenium red and saponin technique. In both the inner and outer ZP layers, stiffness decreased at maturation while, conversely, increased after fertilization. Contextually, at the nanoscale, during maturation both ZP layers displayed a fine filaments network whose length increased while thickness decreased. After fertilization, filaments partially recovered the immature features, appearing again shorter and thicker. Overall, the observed biomechanical modifications were substantiated by ultrastructural findings in the ZP filament mesh. In fertilized ZP, the calculated force necessary to displace ZP filaments resulted quite similar to that previously reported as generated by bovine sperm flagellum. Therefore, in bovine IVF biomechanical modifications of ZP appear ineffective in hindering sperm transit, highlighting the relevance of additional mechanisms operating in vivo.     

The mitochondrial inner membrane protein mitofilin controls cristae morphology

Mitochondria are complex organelles with a highly dynamic distribution and internal organization. Here, we demonstrate that mitofilin, a previously identified mitochondrial protein of unknown function, controls mitochondrial cristae morphology. Mitofilin is enriched in the narrow space between the inner boundary and the outer membranes, where it forms a homotypic interaction and assembles into a large multimeric protein complex. Down-regulation of mitofilin in HeLa cells by using specific small interfering RNA lead to decreased cellular proliferation and increased apoptosis, suggesting abnormal mitochondrial function. Although gross mitochondrial fission and fusion seemed normal, ultrastructural studies revealed disorganized mitochondrial inner membrane. Inner membranes failed to form tubular or vesicular cristae and showed as closely packed stacks of membrane sheets that fused intermittently, resulting in a complex maze of membranous network. Electron microscopic tomography estimated a substantial increase in inner:outer membrane ratio, whereas no cristae junctions were detected. In addition, mitochondria subsequently exhibited increased reactive oxygen species production and membrane potential. Although metabolic flux increased due to mitofilin deficiency, mitochondrial oxidative phosphorylation was not increased accordingly. We propose that mitofilin is a critical organizer of the mitochondrial cristae morphology and thus indispensable for normal mitochondrial function.     

Association of a 66 kDa homolog of Chlamydomonas DC2, a subunit of the outer arm docking complex, with outer arm dynein of sperm flagella in the ascidian Ciona intestinalis

We previously identified a 66 kDa axonemal protein (Ci-Axp66.0) in sperm of the ascidian Ciona intestinalis. Here we found that Ci-Axp66.0 shows sequence similarity to the DC2 subunit of the Chlamydomonas outer arm docking complex. Analysis of secondary structure of Ci-Axp66.0 suggested that the N-terminal two-thirds of the molecule is rich in coiled coil structure, as in Chlamydomonas DC2. Immunogold localization revealed that it is located in the vicinity of outer arm dynein. Ci-Axp66.0 was partly extracted from the axonemes by a high salt solution and co-purified with outer arm dynein. This co-purification was not affected by the absence of Mg(2+) in isolation buffer, indicating that Ci-Axp66.0 is associated with outer arm dynein. These results suggest that Ci-Axp66.0 is a component of the outer arm dynein docking complex in the axonemes of Ciona sperm.     

High‐resolution architecture of the outer membrane of the Gram‐negative bacteria Roseobacter denitrificans

The outer membrane of Gram‐negative bacteria protects the cell against bactericidal substances. Passage of nutrients and waste is assured by outer membrane porins, beta‐barrel transmembrane channels. While atomic structures of several porins have been solved, so far little is known on the supramolecular structure of the outer membrane. Here we present the first high‐resolution view of a bacterial outer membrane gently purified maintaining remnants of peptidoglycan on the perisplasmic surface. Atomic force microscope images of outer membrane fragments of the size of ～50% of the bacterial envelope revealed that outer membrane porins are by far more densely packed than previously assumed. Indeed the outer membrane is a molecular sieve rather than a membrane. Porins cover ～70% of the membrane surface and form locally regular lattices. The potential role of exposed aromatic residues in the formation of the supramolecular assembly is discussed. Finally, we present first structural data of the outer membrane porin from the marine Gram‐negative bacteria Roseobacter denitrificans, and we perform a sequence alignment with porins of known structure.     

The fibrous system of the extracellular matrix of Podocoryne carnea and its degradation by the subumbrellar plate endoderm demonstrated by a monoclonal antibody

Homogenized fragments of crude umbrellar material of the hydromedusa Podocoryne carnea was injected into BALB/c mice. The immunization resulted in the isolation of a monoclonal antibody designated 3A1 which specifically binds to fibrils in the mesogloeal extracellular matrix (ECM) of hydromedusae. In vivo, the architecture and the ultrastructure of the fibrous system in the outer mesogloea (outer ECM) of Podocoryne carnea, and its degradation under in vitro conditions have been described by morphological and immunological criteria. In vivo, 120-150 A thick, striated fibrils (with periodicities up to 50 nm) form a threedimensional network which fills in the entire outer ECM. Vertical fibers (up to 150 nm in diameter) penetrate the three-dimensional network and branch at the subumbrellar and the exumbrellar side. The vertical fibers show uniform distribution over the entire outer ECM. The branches impinge on a dense matrix (about 30 nm in thickness) covering the exumbrellar and subumbrellar surface. In vitro, the fibrillar system does not alter in its basic pattern, neither in isolated outer ECM, nor in portions of outer ECM which is either covered by the exumbrella, or which is attached to both: the exumbrella at the outside, and the subumbrellar plate endoderm at the inner side. After removal of the exumbrellar cells in the latter portions, a characteristic pattern of selective degradation of the outer ECM by the endodermal cells is observed. This process involves three distinct steps: an initial extracellular condensation within the ECM fibrillar network, followed by intercellular internalization of the fibrillar elements and subsequent endocytosis of ECM material. The first step immediately follows the removeal procedure of the exumbrellar cells and is completed within minutes. This process cannot be interrupted by dihydrocytochalasin B (H(2)CB). The second step lasts 24-48 hr, is mediated by cell mechanisms, and can be stopped by H(2)CB. The third step is a slow process (of up to 14 days). It involves intercellular degradation of fibrillar material, endocytosis, and completion of digestion within lysosomes.     

Pal Lipoprotein of Escherichia coli Plays a Major Role in Outer Membrane Integrity

The Tol-Pal system of gram-negative bacteria is composed of five proteins. TolA, TolQ, and TolR are inner membrane proteins, TolB is a periplasmic protein, and Pal, the peptidoglycan-associated lipoprotein, is anchored to the outer membrane. In this study, the roles of Pal and major lipoprotein Lpp were compared in Escherichia coli. lpp and tol-pal mutations have previously been found to perturb the outer membrane permeability barrier and to cause the release of periplasmic proteins and the formation of outer membrane vesicles. In this study, we showed that the overproduction of Pal is able to restore the outer membrane integrity of an lpp strain but that overproduced Lpp has no effect in a pal strain. Together with the previously reported observation that overproduced TolA complements an lpp but not a pal strain, these results indicate that the cell envelope integrity is efficiently stabilized by an epistatic Tol-Pal system linking inner and outer membranes. The density of Pal was measured and found to be lower than that of Lpp. However, Pal was present in larger amounts compared to TolA and TolR proteins. The oligomeric state of Pal was determined and a new interaction between Pal and Lpp was demonstrated.