RESUMEN
Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum. The large GTPase Atlastin has recently been shown to drive endoplasmic reticulum membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated membrane fusion, and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel α helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion.
Asunto(s)
Proteínas de Drosophila/química , Retículo Endoplásmico/química , GTP Fosfohidrolasas/química , Proteínas de Unión al GTP/química , Membrana Dobles de Lípidos/química , Fusión de Membrana , Proteínas de la Membrana/química , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Humanos , Membrana Dobles de Lípidos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Estructura Secundaria de ProteínaRESUMEN
Peroxisomes are ubiquitous organelles housing a variety of essential biochemical pathways. Peroxisome dysfunction causes a spectrum of human diseases known as peroxisome biogenesis disorders (PBD). Although much is known regarding the mechanism of peroxisome biogenesis, it is still unclear how peroxisome dysfunction leads to the disease state. Several recent studies have shown that mutations in Drosophila peroxin genes cause phenotypes similar to those seen in humans with PBDs suggesting that Drosophila might be a useful system to model PBDs. We have analyzed the proteome of Drosophila to identify the proteins involved in peroxisomal biogenesis and homeostasis as well as metabolic enzymes that function within the organelle. The subcellular localization of five of these predicted peroxisomal proteins was confirmed. Similar to Caenorhabditis elegans, Drosophila appears to only utilize the peroxisome targeting signal type 1 system for matrix protein import. This work will further our understanding of peroxisomes in Drosophila and add to the usefulness of this emerging model system.
Asunto(s)
Proteínas de Drosophila/análisis , Drosophila melanogaster/metabolismo , Peroxisomas/metabolismo , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Drosophila melanogaster/enzimología , Peroxisomas/química , Peroxisomas/enzimología , Transporte de Proteínas , Proteoma/análisisRESUMEN
The biogenesis and maintenance of the endoplasmic reticulum (ER) requires membrane fusion. ER homotypic fusion is driven by the large GTPase atlastin. Domain analysis of atlastin shows that a conserved region of the C-terminal cytoplasmic tail is absolutely required for fusion activity. Atlastin in adjacent membranes must associate to bring the ER membranes into molecular contact. Drosophila atlastin dimerizes in the presence of GTPγS but is monomeric with GDP or without nucleotide. Oligomerization requires the juxtamembrane middle domain three-helix bundle, as does efficient GTPase activity. A soluble version of the N-terminal cytoplasmic domain that contains the GTPase domain and the middle domain three-helix bundle serves as a potent, concentration-dependent inhibitor of membrane fusion both in vitro and in vivo. However, atlastin domains lacking the middle domain are without effect. GTP-dependent dimerization of atlastin generates an enzymatically active protein that drives membrane fusion after nucleotide hydrolysis and conformational reorganization.
Asunto(s)
Proteínas de Drosophila/fisiología , GTP Fosfohidrolasas/fisiología , Fusión de Membrana/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Dimerización , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Retículo Endoplásmico/fisiología , GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/genética , Nucleótidos de Guanina/metabolismo , Cinética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
A screen for mutations that affect the recruitment of the exocyst to secretory vesicles identified genes encoding clathrin and proteins that associate or colocalize with clathrin at sites of endocytosis. However, no significant colocalization of the exocyst with clathrin was seen, arguing against a direct role in exocyst recruitment. Rather, these components are needed to recycle the exocytic vesicle SNAREs Snc1p and Snc2p from the plasma membrane into new secretory vesicles where they act to recruit the exocyst. We observe a direct interaction between the exocyst subunit Sec6p and the latter half of the SNARE motif of Snc2p. An snc2 mutation that specifically disrupts this interaction led to exocyst mislocalization and a block in exocytosis in vivo without affecting liposome fusion in vitro. Overexpression of Sec4p partially suppressed the exocyst localization defects of mutations in clathrin and clathrin-associated components. We propose that the exocyst is recruited to secretory vesicles by the combinatorial signals of Sec4-GTP and the Snc proteins. This could help to confer both specificity and directionality to vesicular traffic.
Asunto(s)
Exocitosis , Proteínas R-SNARE/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vesículas Secretoras/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Sitios de Unión , Unión ProteicaRESUMEN
BACKGROUND: Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes. METHODOLOGY/PRINCIPAL FINDINGS: Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex. CONCLUSION/SIGNIFICANCE: Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.
Asunto(s)
Fusión de Membrana/fisiología , Proteínas Munc18/metabolismo , Proteínas Qa-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteína 2 de Membrana Asociada a Vesículas/metabolismo , Animales , Membrana Celular/metabolismo , Humanos , Ratas , Transducción de SeñalRESUMEN
BACKGROUND: Bacillus spores are notoriously resistant to unfavorable conditions such as UV radiation, gamma-radiation, H2O2, desiccation, chemical disinfection, or starvation. Bacillus pumilus SAFR-032 survives standard decontamination procedures of the Jet Propulsion Lab spacecraft assembly facility, and both spores and vegetative cells of this strain exhibit elevated resistance to UV radiation and H2O2 compared to other Bacillus species. PRINCIPAL FINDINGS: The genome of B. pumilus SAFR-032 was sequenced and annotated. Lists of genes relevant to DNA repair and the oxidative stress response were generated and compared to B. subtilis and B. licheniformis. Differences in conservation of genes, gene order, and protein sequences are highlighted because they potentially explain the extreme resistance phenotype of B. pumilus. The B. pumilus genome includes genes not found in B. subtilis or B. licheniformis and conserved genes with sequence divergence, but paradoxically lacks several genes that function in UV or H2O2 resistance in other Bacillus species. SIGNIFICANCE: This study identifies several candidate genes for further research into UV and H2O2 resistance. These findings will help explain the resistance of B. pumilus and are applicable to understanding sterilization survival strategies of microbes.