Melanie D. Ohi

Proteomics Analysis Reveals Stable Multiprotein Complexes in Both Fission and Budding Yeasts Containing Myb-Related Cdc5p/Cef1p, Novel Pre-mRNA Splicing Factors, and snRNAs

ABSTRACT Schizosaccharomyces pombe Cdc5p and its Saccharomyces cerevisiae ortholog, Cef1p, are essential Myb-related proteins implicated in pre-mRNA splicing and contained within large multiprotein complexes. Here we describe the tandem affinity purification (TAP) of Cdc5p- and Cef1p-associated complexes. Using transmission electron microscopy, we show that the purified Cdc5p complex is a discrete structure. The components of the S. pombe Cdc5p/S. cerevisiae Cef1p complexes (termed Cwfs or Cwcs, respectively) were identified using direct analysis of large protein complex (DALPC) mass spectrometry (A. J. Link et al., Nat. Biotechnol. 17:676-682, 1999). At least 26 proteins were detected in the Cdc5p/Cef1p complexes. Comparison of the polypeptides identified by S. pombe Cdc5p purification with those identified by S. cerevisiae Cef1p purification indicates that these two yeast complexes are nearly identical in composition. The majority of S. pombe Cwf proteins and S. cerevisiae Cwc proteins are known pre-mRNA splicing factors including core Sm and U2 and U5 snRNP components. In addition, the complex contains the U2, U5, and U6 snRNAs. Previously uncharacterized proteins were also identified, and we provide evidence that several of these novel factors are involved in pre-mRNA splicing. Our data represent the first comprehensive analysis of CDC5-associated proteins in yeasts, describe a discrete highly conserved complex containing novel pre-mRNA splicing factors, and demonstrate the power of DALPC for identification of components in multiprotein complexes.

Characterization of interactions among the Cef1p-Prp19p-associated splicing complex.

Schizosaccharomyces pombe (Sp) Cdc5p and its Saccharomyces cerevisiae (Sc) ortholog, Cef1p, are essential components of the spliceosome. In S. cerevisiae, a subcomplex of the spliceosome that includes Cef1p can be isolated on its own; this has been termed the nineteen complex (Ntc) because it contains Prp19p. Components of the Ntc include Cef1p, Snt309p, Syf2p/Ntc31p, Ntc30p/lsy1p, Ntc20p and at least six unidentified proteins. We recently identified approximately 30 proteins that copurified with Cdc5p and Cef1p. Previously unidentified S. pombe proteins in this purification were called Cwfs for complexed with five and novel S. cerevisiae proteins were called Cwcs for complexed with Cef1p. Using these proteomics data coupled with available information regarding Ntc composition, we have investigated protein identities and interactions among Ntc components. Our data indicate that Cwc2p, Prp46p, Clf1p, and Syf1p most likely represent Ntc40p, Ntc50p, Ntc77p, and Ntc90p, respectively. We show that Sc Cwc2p interacts with Prp19p and is involved in pre-mRNA splicing. Sp cwf2+, the homolog of Sc CWC2, is allelic with the previously identified Sp prp3+. We present evidence that Sp Cwf7p, an essential protein with obvious homologs in many eukaryotes but not S. cerevisiae, is a functional counterpart of Sc Snt309p and binds Sp Cwf8p (a homolog of Sc Prp19p). Further, our data indicate that a mutation in the U-box of Prp19p disrupts these numerous protein interactions causing Cef1p degradation and Ntc instability.

Structural organization of the functional domains of Clostridium difficile toxins A and B

Clostridium difficile toxins A and B are members of an important class of virulence factors known as large clostridial toxins (LCTs). Toxin action involves four major steps: receptor-mediated endocytosis, translocation of a catalytic glucosyltransferase domain across the membrane, release of the enzymatic moiety by autoproteolytic processing, and a glucosyltransferase-dependent inactivation of Rho family proteins. We have imaged toxin A (TcdA) and toxin B (TcdB) holotoxins by negative stain electron microscopy to show that these molecules are similar in structure. We then determined a 3D structure for TcdA and mapped the organization of its functional domains. The molecule has a “pincher-like” head corresponding to the delivery domain and two tails, long and short, corresponding to the receptor-binding and glucosyltransferase domains, respectively. A second structure, obtained at the acidic pH of an endosome, reveals a significant structural change in the delivery and glucosyltransferase domains, and thus provides a framework for understanding the molecular mechanism of LCT cellular intoxication.

Dephosphorylation of F-BAR protein Cdc15 modulates its conformation and stimulates its scaffolding activity at the cell division site

Cytokinesis in Schizosaccharomyces pombe requires the function of Cdc15, the founding member of the pombe cdc15 homology (PCH) family of proteins. As an early, abundant contractile ring component with multiple binding partners, Cdc15 plays a key role in organizing the ring. We demonstrate that Cdc15 phosphorylation at many sites generates a closed conformation, inhibits Cdc15 assembly at the division site in interphase, and precludes interaction of Cdc15 with its binding partners. Cdc15 dephosphorylation induces an open conformation, oligomerization, and scaffolding activity during mitosis. Cdc15 mutants with reduced phosphorylation precociously appear at the division site in filament-like structures and display increased association with protein partners and the membrane. Our results indicate that Cdc15 phosphoregulation impels both assembly and disassembly of the contractile apparatus and suggest a regulatory strategy that PCH family and BAR superfamily members might broadly employ to achieve temporal specificity in their roles as linkers between membrane and cytoskeleton.

Molecular assembly of botulinum neurotoxin progenitor complexes

Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum and associates with nontoxic neurotoxin-associated proteins to form high-molecular weight progenitor complexes (PCs). The PCs are required for the oral toxicity of BoNT in the context of food-borne botulism and are thought to protect BoNT from destruction in the gastrointestinal tract and aid in absorption from the gut lumen. The PC can differ in size and protein content depending on the C. botulinum strain. The oral toxicity of the BoNT PC increases as the size of the PC increases, but the molecular architecture of these large complexes and how they contribute to BoNT toxicity have not been elucidated. We have generated 2D images of PCs from strains producing BoNT serotypes A1, B, and E using negative stain electron microscopy and single-particle averaging. The BoNT/A1 and BoNT/B PCs were observed as ovoid-shaped bodies with three appendages, whereas the BoNT/E PC was observed as an ovoid body. Both the BoNT/A1 and BoNT/B PCs showed significant flexibility, and the BoNT/B PC was documented as a heterogeneous population of assembly/disassembly intermediates. We have also determined 3D structures for each serotype using the random conical tilt approach. Crystal structures of the individual proteins were placed into the BoNT/A1 and BoNT/B PC electron density maps to generate unique detailed models of the BoNT PCs. The structures highlight an effective platform that can be engineered for the development of mucosal vaccines and the intestinal absorption of oral biologics.

Gle1 functions during mRNA export in an oligomeric complex that is altered in human disease.

The conserved multifunctional protein Gle1 regulates gene expression at multiple steps: nuclear mRNA export, translation initiation, and translation termination. A GLE1 mutation (FinMajor) is causally linked to human lethal congenital contracture syndrome-1 (LCCS1); however, the resulting perturbations on Gle1 molecular function were unknown. FinMajor results in a proline-phenylalanine-glutamine peptide insertion within the uncharacterized Gle1 coiled-coil domain. Here, we find that Gle1 self-associates both in vitro and in living cells via the coiled-coil domain. Electron microscopy reveals that high-molecular-mass Gle1 oligomers form ?26 nm diameter disk-shaped particles. With the Gle1-FinMajor protein, these particles are malformed. Moreover, functional assays document a specific requirement for proper Gle1 oligomerization during mRNA export, but not for Gle1s roles in translation. These results identify a mechanistic step in Gle1s mRNA export function at nuclear pore complexes and directly implicate altered export in LCCS1 disease pathology.

The Human Metapneumovirus Fusion Protein Mediates Entry Via an Interaction with RGD-binding Integrins

ABSTRACT Paramyxoviruses use a specialized fusion protein to merge the viral envelope with cell membranes and initiate infection. Most paramyxoviruses require the interaction of two viral proteins to enter cells; an attachment protein binds cell surface receptors, leading to the activation of a fusion (F) protein that fuses the viral envelope and host cell plasma membrane. In contrast, human metapneumovirus (HMPV) expressing only the F protein is replication competent, suggesting a primary role for HMPV F in attachment and fusion. We previously identified an invariant arginine-glycine-aspartate (RGD) motif in the HMPV F protein and showed that the RGD-binding integrin αVβ1-promoted HMPV infection. Here we show that both HMPV F-mediated binding and virus entry depend upon multiple RGD-binding integrins and that HMPV F can mediate binding and fusion in the absence of the viral attachment (G) protein. The invariant F-RGD motif is critical for infection, as an F-RAE virus was profoundly impaired. Further, F-integrin binding is required for productive viral RNA transcription, indicating that RGD-binding integrins serve as receptors for the HMPV fusion protein. Thus, HMPV F is triggered to induce virus-cell fusion by interactions with cellular receptors in a manner that is independent of the viral G protein. These results suggest a stepwise mechanism of HMPV entry mediated by the F protein through its interactions with cellular receptors, including RGD-binding integrins.

Systematic two-hybrid and comparative proteomic analyses reveal novel yeast pre-mRNA splicing factors connected to Prp19

Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1∶1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.

Molecular and Structural Analysis of the Helicobacter pylori cag Type IV Secretion System Core Complex

ABSTRACT Bacterial type IV secretion systems (T4SSs) can function to export or import DNA, and can deliver effector proteins into a wide range of target cells. Relatively little is known about the structural organization of T4SSs that secrete effector proteins. In this report, we describe the isolation and analysis of a membrane-spanning core complex from the Helicobacter pylori cag T4SS, which has an important role in the pathogenesis of gastric cancer. We show that this complex contains five H. pylori proteins, CagM, CagT, Cag3, CagX, and CagY, each of which is required for cag T4SS activity. CagX and CagY are orthologous to the VirB9 and VirB10 components of T4SSs in other bacterial species, and the other three Cag proteins are unique to H. pylori. Negative stain single-particle electron microscopy revealed complexes 41 nm in diameter, characterized by a 19-nm-diameter central ring linked to an outer ring by spoke-like linkers. Incomplete complexes formed by Δcag3 or ΔcagT mutants retain the 19-nm-diameter ring but lack an organized outer ring. Immunogold labeling studies confirm that Cag3 is a peripheral component of the complex. The cag T4SS core complex has an overall diameter and structural organization that differ considerably from the corresponding features of conjugative T4SSs. These results highlight specialized features of the H. pylori cag T4SS that are optimized for function in the human gastric mucosal environment. IMPORTANCE Type IV secretion systems (T4SSs) are versatile macromolecular machines that are present in many bacterial species. In this study, we investigated a T4SS found in the bacterium Helicobacter pylori. H. pylori is an important cause of stomach cancer, and the H. pylori T4SS contributes to cancer pathogenesis by mediating entry of CagA (an effector protein regarded as a “bacterial oncoprotein”) into gastric epithelial cells. We isolated and analyzed the membrane-spanning core complex of the H. pylori T4SS and showed that it contains unique proteins unrelated to components of T4SSs in other bacterial species. These results constitute the first structural analysis of the core complex from this important secretion system. Type IV secretion systems (T4SSs) are versatile macromolecular machines that are present in many bacterial species. In this study, we investigated a T4SS found in the bacterium Helicobacter pylori. H. pylori is an important cause of stomach cancer, and the H. pylori T4SS contributes to cancer pathogenesis by mediating entry of CagA (an effector protein regarded as a “bacterial oncoprotein”) into gastric epithelial cells. We isolated and analyzed the membrane-spanning core complex of the H. pylori T4SS and showed that it contains unique proteins unrelated to components of T4SSs in other bacterial species. These results constitute the first structural analysis of the core complex from this important secretion system.

The Chlamydia effector chlamydial outer protein N (CopN) sequesters tubulin and prevents microtubule assembly.

Chlamydia species are obligate intracellular pathogens that utilize a type three secretion system to manipulate host cell processes. Genetic manipulations are currently not possible in Chlamydia, necessitating study of effector proteins in heterologous expression systems and severely complicating efforts to relate molecular strategies used by Chlamydia to the biochemical activities of effector proteins. CopN is a chlamydial type three secretion effector that is essential for virulence. Heterologous expression of CopN in cells results in loss of microtubule spindles and metaphase plate formation and causes mitotic arrest. CopN is a multidomain protein with similarity to type three secretion system “plug” proteins from other organisms but has functionally diverged such that it also functions as an effector protein. We show that CopN binds directly to αβ-tubulin but not to microtubules (MTs). Furthermore, CopN inhibits tubulin polymerization by sequestering free αβ-tubulin, similar to one of the mechanisms utilized by stathmin. Although CopN and stathmin share no detectable sequence identity, both influence MT formation by sequestration of αβ-tubulin. CopN displaces stathmin from preformed stathmin-tubulin complexes, indicating that the proteins bind overlapping sites on tubulin. CopN is the first bacterial effector shown to disrupt MT formation directly. This recognition affords a mechanistic understanding of a strategy Chlamydia species use to manipulate the host cell cycle.