The Neurospora crassa TOB Complex: Analysis of the Topology and Function of Tob38 and Tob37

The TOB or SAM complex is responsible for assembling several proteins into the mitochondrial outer membrane, including all β-barrel proteins. We have identified several forms of the complex in Neurospora crassa. One form contains Tob55, Tob38, and Tob37; another contains these three subunits plus the Mdm10 protein; while additional complexes contain only Tob55. As previously shown for Tob55, both Tob37 and Tob38 are essential for viability of the organism. Mitochondria deficient in Tob37 or Tob38 have reduced ability to assemble β-barrel proteins. The function of two hydrophobic domains in the C-terminal region of the Tob37 protein was investigated. Mutant Tob37 proteins lacking either or both of these regions are able to restore viability to cells lacking the protein. One of the domains was found to anchor the protein to the outer mitochondrial membrane but was not necessary for targeting or association of the protein with mitochondria. Examination of the import properties of mitochondria containing Tob37 with deletions of the hydrophobic domains reveals that the topology of Tob37 may be important for interactions between specific classes of β-barrel precursors and the TOB complex

Multivalent Cyclic RGD Conjugates for Targeted Delivery of Small Interfering RNA

We have designed, synthesized and tested conjugates of chemically modified luciferase siRNA (Luc-siRNA) with bi-, tri- and tetravalent cyclic(arginine-glycine-aspartic) peptides (cRGD) that selectively bind to the αvβ3 integrin. The cellular uptake, subcellular distribution and pharmacological effects of the cRGD conjugated Luc-siRNAs as compared to un-conjugated controls were examined using a luciferase reporter cassette stably transfected into αvβ3 positive M21+ human melanoma cells. The M21+ cells exhibited receptor-mediated uptake of cRGD-siRNA conjugates but not of unconjugated control siRNA. The fluorophore-tagged cRGD-siRNA conjugates were taken up by a caveolar endocytotic route and primarily accumulated in cytosolic vesicles. The bi-, tri- and tetravalent cRGD conjugates were taken up by M21+ cells to approximately the same degree. However, there were notable differences in their pharmacological effectiveness. The tri- and tetravalent versions produced progressive, dose-dependent reductions in luciferase expression, while the bivalent version had little effect. The basis for this divergence of uptake and effect is currently unclear. Nonetheless the high selectivity and substantial ‘knock down’ effects of the multivalent cRGD-siRNA conjugates suggest that this targeting and delivery strategy deserves further exploration

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

Analysis of Mutations in <i>Neurospora crassa</i> ERMES Components Reveals Specific Functions Related to β-Barrel Protein Assembly and Maintenance of Mitochondrial Morphology

<div><p>The endoplasmic reticulum mitochondria encounter structure (ERMES) tethers the ER to mitochondria and contains four structural components: Mmm1, Mdm12, Mdm10, and Mmm2 (Mdm34). The Gem1 protein may play a role in regulating ERMES function. <i>Saccharomyces cerevisiae</i> and <i>Neurospora crassa</i> strains lacking any of Mmm1, Mdm12, or Mdm10 are known to show a variety of phenotypic defects including altered mitochondrial morphology and defects in the assembly of β-barrel proteins into the mitochondrial outer membrane. Here we examine ERMES complex components in <i>N. crassa</i> and show that Mmm1 is an ER membrane protein containing a Cys residue near its N-terminus that is conserved in the class Sordariomycetes. The residue occurs in the ER-lumen domain of the protein and is involved in the formation of disulphide bonds that give rise to Mmm1 dimers. Dimer formation is required for efficient assembly of Tom40 into the TOM complex. However, no effects are seen on porin assembly or mitochondrial morphology. This demonstrates a specificity of function and suggests a direct role for Mmm1 in Tom40 assembly. Mutation of a highly conserved region in the cytosolic domain of Mmm1 results in moderate defects in Tom40 and porin assembly, as well as a slight morphological phenotype. Previous reports have not examined the role of Mmm2 with respect to mitochondrial protein import and assembly. Here we show that absence of Mmm2 affects assembly of β-barrel proteins and that lack of any ERMES structural component results in defects in Tom22 assembly. Loss of <i>N. crassa</i> Gem1 has no effect on the assembly of these proteins but does affect mitochondrial morphology.</p></div

An intact unfolded protein response in Trpt1 knockout mice reveals phylogenic divergence in pathways for RNA ligation

Unconventional mRNA splicing by an endoplasmic reticulum stress-inducible endoribonuclease, IRE1, is conserved in all known eukaryotes. It controls the expression of a transcription factor, Hac1p/XBP-1, that regulates gene expression in the unfolded protein response. In yeast, the RNA fragments generated by Ire1p are ligated by tRNA ligase (Trl1p) in a process that leaves a 2′-PO4 2− at the splice junction, which is subsequently removed by an essential 2′-phosphotransferase, Tpt1p. However, animals, unlike yeast, have two RNA ligation/repair pathways that could potentially rejoin the cleaved Xbp-1 mRNA fragments. We report that inactivation of the Trpt1 gene, encoding the only known mammalian homolog of Tpt1p, eliminates all detectable 2′-phosphotransferase activity from cultured mouse cells but has no measurable effect on spliced Xbp-1 translation. Furthermore, the relative translation rates of tyrosine-rich proteins is unaffected by the Trpt1 genotype, suggesting that the pool of (normally spliced) tRNATyr is fully functional in the Trpt1−/− mouse cells. These observations argue against the presence of a 2′-PO4 2− at the splice junction of ligated RNA molecules in Trpt1−/− cells, and suggest that Xbp-1 and tRNA ligation proceed by distinct pathways in yeast and mammals

Characterization of <i>N. crassa</i> Cys to Ser Mmm1 mutants.

<p>A. The Mmm1-HA protein engages in disulphide bonding. Mitochondria (30 µg) were treated with cracking buffer that either did (+BME) or did not (-BME) contain β-mercaptoethanol. Samples were subjected to SDS-PAGE, transferred to nitrocellulose and analyzed by Western blotting for the indicated proteins. B. Coimmunoprecipitation of two tagged forms of Mmm1 from a heterokaryon. An unforced heterokaryon consisting of strains Mmm1-HA3 and Mmm1-Myc10 (HA/Myc) was constructed as described in the Methods. Mitochondria isolated from the heterokaryon were dissolved and treated with anti-Myc agarose beads. Elutions from the beads, or total mitochondrial proteins (mito load, to monitor the input level of proteins), were electrophoresed, blotted, and immunodecorated with the antibodies indicated on the right. Controls were an untagged wild type NCN251 strain (control), the homokaryotic Mmm1-HA3 strain (HA), and the homokaryotic Mmm1-Myc10 strain (Myc). Arrowheads on the left indicate the relevant bands in panels containing non-specific background bands. C. Tenfold dilutions of conidiaspores from strains expressing control (Mmm1-HA) and mutant HA-tagged versions of Mmm1 were spotted on plates containing Vogel’s sorbose medium. The plates were incubated at 30°C for 48 h and then photographed. D. Strains expressing control (Mmm1-HA) and mutant HA-tagged versions of Mmm1 were grown on solid Vogel’s media, stained with MitoTracker Green FM and examined by confocal fluorescence microscopy. Mitochondria in the <i>Δmmm1</i> strain are shown for comparison. Bar represents 10 µm. E. Western blot analysis of Mmm1 Cys mutant crude mitochondria. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g003" target="_blank">Figure 3A</a>, but mitochondria were only analyzed by non-reducing SDS-PAGE. F. Cell fractionation of the indicated strains as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g001" target="_blank">Figure 1A</a> except that Tom22 was the mitochondrial marker.</p

Characterization of the <i>Δgem1</i> strain.

<p>A. Examination of mitochondrial morphology as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g003" target="_blank">Figure 3D</a>. B. Growth rates as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g003" target="_blank">Figure 3C</a>. C. Steady state levels of mitochondrial proteins as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g005" target="_blank">Figure 5A</a>. D. β-barrel protein (Tom40 and porin) assembly as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g004" target="_blank">Figure 4A and B</a>, respectively. E. Tom22 assembly as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g004" target="_blank">Figure 4C</a>. F. Mitochondrial phospholipid content as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone-0071837-g006" target="_blank">Figure 6G</a>.</p

Alignments of fungal Mmm1 proteins.

<p>A. Alignment of Mmm1 N-terminal regions from several Ascomycetes. Known <i>S. cerevisiae</i> (N50, N55 and N59) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071837#pone.0071837-Stroud1" target="_blank">[12]</a> and potential N-glycosylation sites in other species are highlighted in black. Cys residue conserved in Sordariomycetes is shaded in grey. B. Predicted transmembrane domain of Mmm1is present in most fungi, but absent in Mucormycotina and Chytridiomycota. The predicted <i>N. crassa</i> transmembrane domain is highlighted in grey. Identity/similarity symbols are for the alignment of the Ascomycota and Basidiomycota only. * indicates conserved residues, : indicates conservation of groups with strongly similar properties (score of >0.5 in the Gonnet PAM 250 matrix),. indicates conservation of groups with weakly similar properties (score of <0.5 in the Gonnet PAM 250 matrix). C. Alignment of the highly conserved region of Mmm1 chosen for mutation analysis. The nine amino acid region that was chosen for mutation is highlighted in the <i>N. crassa</i> protein (residues 116-124). Symbols (as in panel B) are for the alignment of all proteins. Abbreviations: N.c., <i>Neurospora crassa</i>; G.z., <i>Gibberella zeae</i>; C.g., <i>Chaetomium globosum</i>; S.m., <i>Sordaria macrospora</i>; M.o., <i>Magnaporthe oryzae</i>; P.a., <i>Podospora anserina</i>; F.o. <i>Fusarium oxysporum</i>; V.d., <i>Verticillium dahliae</i>; A.n., <i>Aspergillus nidulans</i>; T.t.,<i>Trichophyton tonsurans</i>; C.i., <i>Coccidioides immitis</i>; P.b., <i>Paracoccidioides brasiliensis</i>; S.s., <i>Sclerotinia sclerotiorum</i>; B.f., <i>Botryotinia fuckeliana</i>; P.t., <i>Pyrenophora teres</i>; S.c., <i>Saccharomyces cerevisiae</i>; K.l., <i>Kluyveromyces lactis</i>; C.a., <i>Candida albicans</i>; S.p., <i>Schizosaccharomyces pombe</i>; U.m., <i>Ustilago maydis</i>; C.n., <i>Cryptocuccus neoformans</i>; R.o., <i>Rhizopus oryzae</i>; B.d., <i>Batrachochytrium dendrobatidis</i>.</p

Strains used in this study.

1<p>Fungal Genetics Stock Center.</p

Subcellular localization of HA-tagged Mmm1.

<p>A. Cell fractionation by differential and sucrose flotation gradient centrifugation was performed on the indicated strains. Flotation gradient purified mitochondrial, post mitochondrial pellet (PMP) and cytosolic fractions (30 µg) were subjected to SDS-PAGE followed by Western blot analysis using antibodies to the indicated proteins. Kar2, ER marker; Tom40, mitochondrial marker; Arginase, cytosolic marker. B. Outer membrane vesicles (OMVs) were isolated from cells expressing Mmm1-HA protein, subjected to SDS-PAGE and analyzed by Western blot for the presence or absence of the indicated proteins. The leftmost lane contains whole mitochondria C. Cell fractionation and detection of indicated proteins. As in panel A but Tom22 is the mitochondrial marker. D. Flotation gradient purified mitochondria and PMP isolated from the Mmm1-HA strain were treated with 0.1 M sodium carbonate at pH 11.0, 11.5 or 12.0. Membrane sheets were pelleted by ultracentrifugation. Proteins in the supernatant were precipitated with trichloroacetic acid. Pellet (pel) and supernatant (sup) fractions were then subjected to SDS-PAGE, transferred to nitrocellulose and analyzed by Western blot using antibodies to the indicated proteins.</p