The UNI3 Gene Is Required for Assembly of Basal Bodies of Chlamydomonas and Encodes ��-Tubulin, a New Member of the Tubulin Superfamily

We have cloned the UNI3 gene in Chlamydomonas and find that it encodes a new member of the tubulin superfamily. Although Uni3p shares significant sequence identity with ��-, ��-, and ��-tubulins, there is a region of Uni3p that has no similarity to tubulins or other known proteins. Mutant uni3���1 cells assemble zero, one, or two flagella. Pedigree analysis suggests that flagellar number in uni3���1 cells is a function of the age of the cell. The uniflagellate uni3���1 cells show a positional phenotype; the basal body opposite the eyespot templates the single flagellum. A percentage of uni3���1 cells also fail to orient the cleavage furrow properly, and basal bodies have been implicated in the placement of cleavage furrows in Chlamydomonas. Finally when uni3���1 cells are observed by electron microscopy, doublet rather than triplet microtubules are observed at the proximal end of the basal bodies. We propose that the Uni3 tubulin is involved in both the function and cell cycle-dependent maturation of basal bodies/centrioles.

Assembly of a Novel Cartilage Matrix Protein Filamentous Network: Molecular Basis of Differential Requirement of von Willebrand Factor A Domains

Cartilage matrix protein (CMP) is the prototype of the newly discovered matrilin family, all of which contain von Willebrand factor A domains. Although the function of matrilins remain unclear, we have shown that, in primary chondrocyte cultures, CMP (matrilin-1) forms a filamentous network, which is made up of two types of filaments, a collagen-dependent one and a collagen-independent one. In this study, we demonstrate that the collagen-independent CMP filaments are enriched in pericellular compartments, extending directly from chondrocyte membranes. Their morphology can be distinguished from that of collagen filaments by immunogold electron microscopy, and mimicked by that of self-assembled purified CMP. The assembly of CMP filaments can occur from transfection of a wild-type CMP transgene alone in skin fibroblasts, which do not produce endogenous CMP. Conversely, assembly of endogenous CMP filaments by chondrocytes can be inhibited specifically by dominant negative CMP transgenes. The two A domains within CMP serve essential but different functions during network formation. Deletion of the A2 domain converts the trimeric CMP into a mixture of monomers, dimers, and trimers, whereas deletion of the A1 domain does not affect the trimeric configuration. This suggests that the A2 domain modulates multimerization of CMP. Absence of either A domain from CMP abolishes its ability to form collagen-independent filaments. In particular, Asp22 in A1 and Asp255 in A2 are essential; double point mutation of these residues disrupts CMP network formation. These residues are part of the metal ion���dependent adhesion sites, thus a metal ion���dependent adhesion site���mediated adhesion mechanism may be applicable to matrilin assembly. Taken together, our data suggest that CMP is a bridging molecule that connects matrix components in cartilage to form an integrated matrix network.

The Role of Preassembled Cytoplasmic Complexes in Assembly of Flagellar Dynein Subunits

Previous work has revealed a cytoplasmic pool of flagellar precursor proteins capable of contributing to the assembly of new flagella, but how and where these components assemble is unknown. We tested Chlamydomonas outer-dynein arm subunit stability and assembly in the cytoplasm of wild-type cells and 11 outer dynein arm assembly mutant strains (oda1-oda11) by Western blotting of cytoplasmic extracts, or immunoprecipitates from these extracts, with five outer-row dynein subunit-specific antibodies. Western blots reveal that at least three oda mutants (oda6, oda7, and oda9) alter the level of a subunit that is not the mutant gene product. Immunoprecipitation shows that large preassembled flagellar complexes containing all five tested subunits (three heavy chains and two intermediate chains) exist within wild-type cytoplasm. When the preassembly of these subunits was examined in oda strains, we observed three patterns: complete coassembly (oda 1, 3, 5, 8, and 10), partial coassembly (oda7 and oda11), and no coassembly (oda2, 6, and 9) of the four tested subunits with HC��. Our data, together with previous studies, suggest that flagellar outer-dynein arms preassemble into a complete Mr ��� 2 �� 106 dynein arm that resides in a cytoplasmic precursor pool before transport into the flagellar compartment.

Characterization of the Golgi Complex Cleared of Proteins in Transit and Examination of Calcium Uptake Activities

To characterize endogenous molecules and activities of the Golgi complex, proteins in transit were >99% cleared from rat hepatocytes by using cycloheximide (CHX) treatment. The loss of proteins in transit resulted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that >90% of the elements could be positively identified as Golgi stacks or cisternae. Biochemical analysis showed that the cis-, medial-, trans-, and TGN Golgi markers were enriched over the postnuclear supernatant 200- to 400-fold with and 400- to 700-fold without proteins in transit. To provide information on a mechanism for import of calcium required at the later stages of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a thapsigargin-resistant pump not resident to the Golgi complex and a thapsigargin-sensitive pump resident to the Golgi. Experiments using CHX SGF1 showed that the thapsigargin-resistant activity was a plasma membrane calcium ATPase isoform in transit to the plasma membrane and the thapsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum calcium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.

Initiation of Assembly of the Cell Envelope Barrier Structure of Stratified Squamous Epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.

Assembly of the Nuclear Transcription and Processing Machinery: Cajal Bodies (Coiled Bodies) and Transcriptosomes

We have examined the distribution of RNA transcription and processing factors in the amphibian oocyte nucleus or germinal vesicle. RNA polymerase I (pol I), pol II, and pol III occur in the Cajal bodies (coiled bodies) along with various components required for transcription and processing of the three classes of nuclear transcripts: mRNA, rRNA, and pol III transcripts. Among these components are transcription factor IIF (TFIIF), TFIIS, splicing factors, the U7 small nuclear ribonucleoprotein particle, the stem���loop binding protein, SR proteins, cleavage and polyadenylation factors, small nucleolar RNAs, nucleolar proteins that are probably involved in pre-rRNA processing, and TFIIIA. Earlier studies and data presented here show that several of these components are first targeted to Cajal bodies when injected into the oocyte and only subsequently appear in the chromosomes or nucleoli, where transcription itself occurs. We suggest that pol I, pol II, and pol III transcription and processing components are preassembled in Cajal bodies before transport to the chromosomes and nucleoli. Most components of the pol II transcription and processing pathway that occur in Cajal bodies are also found in the many hundreds of B-snurposomes in the germinal vesicle. Electron microscopic images show that B-snurposomes consist primarily, if not exclusively, of 20- to 30-nm particles, which closely resemble the interchromatin granules described from sections of somatic nuclei. We suggest the name pol II transcriptosome for these particles to emphasize their content of factors involved in synthesis and processing of mRNA transcripts. We present a model in which pol I, pol II, and pol III transcriptosomes are assembled in the Cajal bodies before export to the nucleolus (pol I), to the B-snurposomes and eventually to the chromosomes (pol II), and directly to the chromosomes (pol III). The key feature of this model is the preassembly of the transcription and processing machinery into unitary particles. An analogy can be made between ribosomes and transcriptosomes, ribosomes being unitary particles involved in translation and transcriptosomes being unitary particles for transcription and processing of RNA.

Assembly of �� protein into Alzheimer paired helical filaments depends on a local sequence motif (306VQIVYK311) forming �� structure

We have searched for a minimal interaction motif in �� protein that supports the aggregation into Alzheimer-like paired helical filaments. Digestion of the repeat domain with different proteases yields a GluC-induced fragment comprising 43 residues (termed PHF43), which represents the third repeat of �� plus some flanking residues. This fragment self assembles readily into thin filaments without a paired helical appearance, but these filaments are highly competent to nucleate bona fide PHFs from full-length ��. Probing the interactions of PHF43 with overlapping peptides derived from the full �� sequence yields a minimal hexapeptide interaction motif of 306VQIVYK311 at the beginning of the third internal repeat. This motif coincides with the highest predicted ��-structure potential in ��. CD and Fourier transform infrared spectroscopy shows that PHF43 acquires pronounced �� structure in conditions of self assembly. Point mutations in the hexapeptide region by proline-scanning mutagenesis prevent the aggregation. The data indicate that PHF assembly is initiated by a short fragment containing the minimal interaction motif forming a local �� structure embedded in a largely random-coil protein.

Archimedean solids: Transition metal mediated rational self-assembly of supramolecular-truncated tetrahedra

A family of nanoscale-sized supramolecular cage compounds with a polyhedral framework is prepared by self-assembly from tritopic building blocks and rectangular corner units via noncovalent coordination interactions. These highly symmetrical cage compounds are described as face-directed, self-assembled truncated tetrahedra with Td symmetry.

Self-assembly of large, ordered lamellae from non-bilayer lipids and integral membrane proteins in vitro

In many biological membranes, the major lipids are ���non-bilayer lipids,��� which in purified form cannot be arranged in a lamellar structure. The structural and functional roles of these lipids are poorly understood. This work demonstrates that the in vitro association of the two main components of a membrane, the non-bilayer lipid monogalactosyldiacylglycerol (MGDG) and the chlorophyll-a/b light-harvesting antenna protein of photosystem II (LHCII) of pea thylakoids, leads to the formation of large, ordered lamellar structures: (i) thin-section electron microscopy and circular dichroism spectroscopy reveal that the addition of MGDG induces the transformation of isolated, disordered macroaggregates of LHCII into stacked lamellar aggregates with a long-range chiral order of the complexes; (ii) small-angle x-ray scattering discloses that LHCII perturbs the structure of the pure lipid and destroys the inverted hexagonal phase; and (iii) an analysis of electron micrographs of negatively stained 2D crystals indicates that in MGDG-LHCII the complexes are found in an ordered macroarray. It is proposed that, by limiting the space available for MGDG in the macroaggregate, LHCII inhibits formation of the inverted hexagonal phase of lipids; in thylakoids, a spatial limitation is likely to be imposed by the high concentration of membrane-associated proteins.

Pentameric assembly of a neuronal glutamate transporter

Freeze-fracture electron microscopy was used to study the structure of a human neuronal glutamate transporter (EAAT3). EAAT3 was expressed in Xenopus laevis oocytes, and its function was correlated with the total number of transporters in the plasma membrane of the same cells. Function was assayed as the maximum charge moved in response to a series of transmembrane voltage pulses. The number of transporters in the plasma membrane was determined from the density of a distinct 10-nm freeze-fracture particle, which appeared in the protoplasmic face only after EAAT3 expression. The linear correlation between EAAT3 maximum carrier-mediated charge and the total number of the 10-nm particles suggested that this particle represented functional EAAT3 in the plasma membrane. The cross-sectional area of EAAT3 in the plasma membrane (48 �� 5 nm2) predicted 35 �� 3 transmembrane ��-helices in the transporter complex. This information along with secondary structure models (6���10 transmembrane ��-helices) suggested an oligomeric state for EAAT3. EAAT3 particles were pentagonal in shape in which five domains could be identified. They exhibited fivefold symmetry because they appeared as equilateral pentagons and the angle at the vertices was 110��. Each domain appeared to contribute to an extracellular mass that projects ���3 nm into the extracellular space. Projections from all five domains taper toward an axis passing through the center of the pentagon, giving the transporter complex the appearance of a penton-based pyramid. The pentameric structure of EAAT3 offers new insights into its function as both a glutamate transporter and a glutamate-gated chloride channel.

Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system

Many bacterial pathogens of plants and animals have evolved a specialized protein-secretion system termed type III to deliver bacterial proteins into host cells. These proteins stimulate or interfere with host cellular functions for the pathogen's benefit. The Salmonella typhimurium pathogenicity island 1 encodes one of these systems that mediates this bacterium's ability to enter nonphagocytic cells. Several components of this type III secretion system are organized in a supramolecular structure termed the needle complex. This structure is made of discrete substructures including a base that spans both membranes and a needle-like projection that extends outward from the bacterial surface. We demonstrate here that the type III secretion export apparatus is required for the assembly of the needle substructure but is dispensable for the assembly of the base. We show that the length of the needle segment is determined by the type III secretion associated protein InvJ. We report that InvG, PrgH, and PrgK constitute the base and that PrgI is the main component of the needle of the type III secretion complex. PrgI homologs are present in type III secretion systems from bacteria pathogenic for animals but are absent from bacteria pathogenic for plants. We hypothesize that the needle component may establish the specificity of type III secretion systems in delivering proteins into either plant or animal cells.

ATP-dependent Membrane Assembly of F-Actin Facilitates Membrane Fusion

We recently established an in vitro assay that monitors the fusion between latex-bead phagosomes and endocytic organelles in the presence of J774 macrophage cytosol (Jahraus et al., 1998). Here, we show that different reagents affecting the actin cytoskeleton can either inhibit or stimulate this fusion process. Because the membranes of purified phagosomes can assemble F-actin de novo from pure actin with ATP (Defacque et al., 2000a), we focused here on the ability of membranes to nucleate actin in the presence of J774 cytosolic extracts. For this, we used F-actin sedimentation, pyrene actin assays, and torsional rheometry, a biophysical approach that could provide kinetic information on actin polymerization and gel formation. We make two major conclusions. First, under our standard in vitro conditions (4 mg/ml cytosol and 1 mM ATP), the presence of membranes actively catalyzed the assembly of cytosolic F-actin, which assembled into highly viscoelastic gels. A model is discussed that links these results to how the actin may facilitate fusion. Second, cytosolic actin paradoxically polymerized more under ATP depletion than under high-ATP conditions, even in the absence of membranes; we discuss these data in the context of the well described, large increases in F-actin seen in many cells during ischemia.

Assembly of the neutrophil respiratory burst oxidase: A direct interaction between p67PHOX and cytochrome b558

Activation of the phagocyte NADPH oxidase complex requires the assembly of the cytosolic factors p47PHOX, p67PHOX, p40PHOX, and Rac1 or Rac2, with the membrane-bound cytochrome b558. Whereas the interaction of p47PHOX with cytochrome b558 is well established, an interaction between p67PHOX and cytochrome b558 has never been investigated. We report here a direct interaction between p67PHOX and cytochrome b558. First, labeled p67PHOX recognizes a 91-kDa band in specific granules from a normal patient but not from a cytochrome b558-deficient patient. Second, p67PHOX binds to cytochrome b558 that has been bound to nitrocellulose. Third, GTP-p67PHOX bound to glutathione agarose is able to pull down cytochrome b558. Rac1-GTP or Rac1-GDP increased the binding of p67PHOX to cytochrome b558, suggesting that at least one of the oxidase-related functions of Rac1 is to promote the interaction between p67PHOX and cytochrome b558.

Identification of two residues in MCM5 critical for the assembly of MCM complexes and Stat1-mediated transcription activation in response to IFN-��

In response to IFN-��, the latent cytoplasmic Stat1 (signal transducer and activator of transcription) proteins translocate into the nucleus and activate transcription. We showed previously that Stat1 recruits a group of nuclear proteins, among them MCM5 (minichromosome maintenance) and MCM3, for transcription activation. MCM5 directly interacts with the transcription activation domain (TAD) of Stat1 and enhances Stat1-mediated transcription activation. In this report, we identified two specific residues (R732, K734) in MCM5 that are required for the direct interaction between Stat1 and MCM5 both in vitro and in vivo. MCM5 containing mutations of R732/K734 did not enhance Stat1-mediated transcription activation in response to IFN-��. In addition, it also failed to form complexes with other MCM proteins in vivo, suggesting that these two residues may be important for an interaction domain in MCM5. Furthermore, MCM5 bearing mutations in its ATPase and helicase domains did not enhance Stat1 activity. In vitro binding assays indicate that MCM3 does not interact directly with Stat1, suggesting that the presence of MCM3 in the group of Stat1TAD-interacting proteins is due to the association of MCM3 with MCM5. Finally, gel filtration analyses of nuclear extracts from INF-��-treated cells demonstrate that there is a MCM5/3 subcomplex coeluting with Stat1. Together, these results strongly suggest that Stat1 recruits a MCM5/3 subcomplex through direct interaction with MCM5 in the process of IFN-��-induced gene activation.