scholarly journals Interaction Maps of the Saccharomyces cerevisiae ESCRT-III Protein Snf7

2013 ◽  
Vol 12 (11) ◽  
pp. 1538-1546 ◽  
Author(s):  
Barbara Sciskala ◽  
Ralf Kölling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Interaction Network ◽  
Multivesicular Body ◽  
Binding Motifs ◽  
Content Type ◽  
Endosomal Membrane ◽  
Binding Partners ◽  
First Time ◽  
Complementary Binding

ABSTRACT The Saccharomyces cerevisiae ESCRT-III protein Snf7 is part of an intricate interaction network at the endosomal membrane. Interaction maps of Snf7 were established by measuring the degree of binding of individual binding partners to putative binding motifs along the Snf7 sequence by glutathione S -transferase (GST) pulldown. For each interaction partner, distinct binding profiles were obtained. The following observations were made. The ESCRT-III subunits Vps20 and Vps24 showed a complementary binding pattern, suggesting a model for the series of events in the ESCRT-III functional cycle. Vps4 bound to individual Snf7 motifs but not to full-length Snf7. This suggests that Vps4 does not bind to the closed conformation of Snf7. We also demonstrate for the first time that the ALIX/Bro1 homologue Rim20 binds to the α6 helix of Snf7. Analysis of a Snf7 α6 deletion mutant showed that the α6 helix is crucial for binding of Bro1 and Rim20 in vivo and is indispensable for the multivesicular body (MVB)-sorting and Rim-signaling functions of Snf7. The Snf7Δα6 protein still appeared to be incorporated into ESCRT-III complexes at the endosomal membrane, but disassembly of the complex seemed to be defective. In summary, our study argues against the view that the ESCRT cycle is governed by single one-to-one interactions between individual components and emphasizes the network character of the ESCRT interactions.

scholarly journals Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
Author(s):  
Fabien Lefèbvre ◽  
Valérie Prouzet-Mauléon ◽  
Michel Hugues ◽  
Marc Crouzet ◽  
Aurélie Vieillemard ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Trafficking ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Rho Gtpase ◽  
Secretory Vesicles ◽  
Gtpase Activating Protein ◽  
Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

scholarly journals Evidence ofIn VivoProphage Induction during Clostridium difficile Infection

2012 ◽  
Vol 78 (21) ◽  
pp. 7662-7670 ◽  
Author(s):  
Mathieu Meessen-Pinard ◽  
Ognjen Sekulovic ◽  
Louis-Charles Fortier
Keyword(s):  
Clostridium Difficile ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Unique Character ◽  
Viral Particles ◽  
Potential Impact ◽  
First Time ◽  
Culture Supernatants

ABSTRACTProphages contribute to the evolution and virulence of most bacterial pathogens, but their role inClostridium difficileis unclear. Here we describe the isolation of fourMyoviridaephages, ϕMMP01, ϕMMP02, ϕMMP03, and ϕMMP04, that were recovered as free viral particles in the filter-sterilized stool supernatants of patients suffering fromC. difficileinfection (CDI). Furthermore, identical prophages were found in the chromosomes ofC. difficileisolated from the corresponding fecal samples. We therefore provide, for the first time, evidence ofin vivoprophage induction during CDI. We completely sequenced the genomes of ϕMMP02 and ϕMMP04, and bioinformatics analyses did not reveal the presence of virulence factors but underlined the unique character of ϕMMP04. We also studied the mobility of ϕMMP02 and ϕMMP04 prophagesin vitro. Both prophages were spontaneously induced, with 4 to 5 log PFU/ml detected in the culture supernatants of the corresponding lysogens. When lysogens were grown in the presence of subinhibitory concentrations of ciprofloxacin, moxifloxacin, levofloxacin, or mitomycin C, the phage titers further increased, reaching 8 to 9 log PFU/ml in the case of ϕMMP04. In summary, our study highlights the extensive genetic diversity and mobility ofC. difficileprophages. Moreover, antibiotics known to represent risk factors for CDI, such as quinolones, can stimulate prophage mobilityin vitroand probablyin vivoas well, which underscores their potential impact on phage-mediated horizontal gene transfer events and the evolution ofC. difficile.

scholarly journals Analysis of Damage Tolerance Pathways in Saccharomyces cerevisiae: a Requirement for Rev3 DNA Polymerase in Translesion Synthesis

1998 ◽  
Vol 18 (2) ◽  
pp. 960-966 ◽  
Author(s):  
K. Baynton ◽  
A. Bresson-Roy ◽  
R. P. P. Fuchs
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Polymerase ◽  
Excision Repair ◽  
Translesion Synthesis ◽  
Hybridization Technique ◽  
Nucleotide Excision ◽  
Yeast Transformants ◽  
Plasmid Design ◽  
First Time

ABSTRACT The replication of double-stranded plasmids containing a singleN-2-acetylaminofluorene (AAF) adduct located in a short, heteroduplex sequence was analyzed in Saccharomyces cerevisiae. The strains used were proficient or deficient for the activity of DNA polymerase ζ (REV3 andrev3Δ, respectively) in a mismatch and nucleotide excision repair-defective background (msh2Δ rad10Δ). The plasmid design enabled the determination of the frequency with which translesion synthesis (TLS) and mechanisms avoiding the adduct by using the undamaged, complementary strand (damage avoidance mechanisms) are invoked to complete replication. To this end, a hybridization technique was implemented to probe plasmid DNA isolated from individual yeast transformants by using short, 32P-end-labeled oligonucleotides specific to each strand of the heteroduplex. In both the REV3 and rev3Δ strains, the two strands of an unmodified heteroduplex plasmid were replicated in ∼80% of the transformants, with the remaining 20% having possibly undergone prereplicative MSH2-independent mismatch repair. However, in the presence of the AAF adduct, TLS occurred in only 8% of theREV3 transformants, among which 97% was mostly error free and only 3% resulted in a mutation. All TLS observed in theREV3 strain was abolished in the rev3Δ mutant, providing for the first time in vivo biochemical evidence of a requirement for the Rev3 protein in TLS.

scholarly journals Coordinated Hsp110 and Hsp104 Activities Power Protein Disaggregation in Saccharomyces cerevisiae

2017 ◽  
Vol 37 (11) ◽  
Author(s):  
Jayasankar Mohanakrishnan Kaimal ◽  
Ganapathi Kandasamy ◽  
Fabian Gasser ◽  
Claes Andréasson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Protein Aggregation ◽  
Yeast Cells ◽  
Eukaryotic Cells ◽  
Content Type ◽  
Dependent Protein ◽  
And Function ◽  
Cellular Dysfunction

ABSTRACT Protein aggregation is intimately associated with cellular stress and is accelerated during aging, disease, and cellular dysfunction. Yeast cells rely on the ATP-consuming chaperone Hsp104 to disaggregate proteins together with Hsp70. Hsp110s are ancient and abundant chaperones that form complexes with Hsp70. Here we provide in vivo data showing that the Saccharomyces cerevisiae Hsp110s Sse1 and Sse2 are essential for Hsp104-dependent protein disaggregation. Following heat shock, complexes of Hsp110 and Hsp70 are recruited to protein aggregates and function together with Hsp104 in the disaggregation process. In the absence of Hsp110, targeting of Hsp70 and Hsp104 to the aggregates is impaired, and the residual Hsp104 that still reaches the aggregates fails to disaggregate. Thus, coordinated activities of both Hsp104 and Hsp110 are required to reactivate aggregated proteins. These findings have important implications for the understanding of how eukaryotic cells manage misfolded and amyloid proteins.

scholarly journals Structure and function of human Vps20 and Snf7 proteins

2004 ◽  
Vol 377 (3) ◽  
pp. 693-700 ◽  
Author(s):  
Jeremy W. PECK ◽  
Emma T. BOWDEN ◽  
Peter D. BURBELO
Keyword(s):  
Protein Interactions ◽  
Osmotic Shock ◽  
Multivesicular Body ◽  
Protein Protein Interactions ◽  
Interacting Protein ◽  
Endosomal Membrane ◽  
Genomic Studies ◽  
Vacuolar Protein ◽  
And Function

Snf7p (sucrose non-fermenting) and Vps20p (vacuolar protein-sorting) are small coil-coiled proteins involved in yeast MVB (multivesicular body) structure, formation and function. In the present study, we report the identification of three human homologues of yeast Snf7p, designated hSnf7-1, hSnf7-2 and hSnf7-3, and a single human Vps20p homologue, designated hVps20, that may have similar roles in humans. Immunofluorescence studies showed that hSnf7-1 and hSnf7-3 localized in large vesicular structures that also co-localized with late endosomal/lysosomal structures induced by overexpressing an ATPase-defective Vps4-A mutant. In contrast, overexpressed hVps20 showed a typical endosomal membrane-staining pattern, and co-expression of hVps20 with Snf7-1 dispersed the large Snf7-staining vesicles. Interestingly, overexpression of both hSnf7 and hVps20 proteins induced a post-endosomal defect in cholesterol sorting. To explore possible protein–protein interactions involving hSnf7 proteins, we used information from yeast genomic studies showing that yeast Snf7p can interact with proteins involved in MVB function. Using a glutathione S-transferase-capture approach with several mammalian homologues of such yeast Snf7p-interacting proteins, we found that all three hSnf7s interacted with mouse AIP1 [ALG-2 (apoptosis-linked gene 2) interacting protein 1], a mammalian Bro1p [BCK1 (bypass of C kinase)-like resistance to osmotic shock]-containing protein involved in cellular vacuolization and apoptosis. Whereas mapping experiments showed that the N-terminus of AIP1 containing both a Bro1 and an α-helical domain were required for interaction with hSnf7-1, Snf7-1 did not interact with another human Bro1-containing molecule, rhophilin-2. Co-immunoprecipitation experiments confirmed the in vivo interaction of hSnf7-1 and AIP1. Additional immunofluorescence experiments showed that hSnf7-1 recruited cytosolic AIP1 to the Snf7-induced vacuolar-like structures. Together these results suggest that mammalian Vps20, AIP1 and Snf7 proteins, like their yeast counterparts, play roles in MVB function.

scholarly journals The Stationary-Phase Cells of Saccharomyces cerevisiae Display Dynamic Actin Filaments Required for Processes Extending Chronological Life Span

2015 ◽  
Vol 35 (22) ◽  
pp. 3892-3908 ◽  
Author(s):  
Pavla Vasicova ◽  
Renata Lejskova ◽  
Ivana Malcova ◽  
Jiri Hasek
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Stationary Phase ◽  
Actin Filaments ◽  
Phase Cell ◽  
Content Type ◽  
Chronological Aging ◽  
Yeast Cultures ◽  
Actin Cables ◽  
Mitochondrial Networks

Stationary-growth-phaseSaccharomyces cerevisiaeyeast cultures consist of nondividing cells that undergo chronological aging. For their successful survival, the turnover of proteins and organelles, ensured by autophagy and the activation of mitochondria, is performed. Some of these processes are engaged in by the actin cytoskeleton. InS. cerevisiaestationary-phase cells, F actin has been shown to form static aggregates named actin bodies, subsequently cited to be markers of quiescence. Ourin vivoanalyses revealed that stationary-phase cultures contain cells with dynamic actin filaments, besides the cells with static actin bodies. The cells with dynamic actin displayed active endocytosis and autophagy and well-developed mitochondrial networks. Even more, stationary-phase cell cultures grown under calorie restriction predominantly contained cells with actin cables, confirming that the presence of actin cables is linked to successful adaptation to stationary phase. Cells with actin bodies were inactive in endocytosis and autophagy and displayed aberrations in mitochondrial networks. Notably, cells of the respiratory activity-deficientcox4Δ strain displayed the same mitochondrial aberrations and actin bodies only. Additionally, our results indicate that mitochondrial dysfunction precedes the formation of actin bodies and the appearance of actin bodies corresponds to decreased cell fitness. We conclude that the F-actin status reflects the extent of damage that arises from exponential growth.

scholarly journals Antiviral Activities of Different Interferon Types and Subtypes against Hepatitis E Virus Replication

2016 ◽  
Vol 60 (4) ◽  
pp. 2132-2139 ◽  
Author(s):  
Daniel Todt ◽  
Catherine François ◽  
Anggakusuma ◽  
Patrick Behrendt ◽  
Michael Engelmann ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Type I ◽  
Subgenomic Replicon ◽  
Content Type ◽  
Transplant Patients ◽  
Culture Models ◽  
Antiviral Activities ◽  
First Time

ABSTRACTHepatitis E virus (HEV) is the causative agent of hepatitis E in humans and a member of the genusOrthohepevirusin the familyHepeviridae. HEV infections are the common cause of acute hepatitis but can also take chronic courses. Ribavirin is the treatment of choice for most patients, and type I interferon (IFN) has been evaluated in a few infected transplant patientsin vivo. In this study, the antiviral effects of different exogenously administered interferons were investigated by using state-of-the-art subgenomic replicon and full-length HEV genome cell culture models. Hepatitis C virus (HCV) subgenomic replicons based on the genotype 2a JFH1 isolate served as the reference. The experiments revealed that HEV RNA replication was inhibited by the application of all types of IFN, including IFN-α (type I), IFN-γ (type II), and IFN-λ3 (type III), but to a far lesser extent than HCV replication. Simultaneous determination of interferon-stimulated gene (ISG) expression levels for all IFN types demonstrated efficient downregulation by HEV. Furthermore, different IFN-α subtypes were also able to block viral replication in combination with ribavirin. The IFN-α subtypes 2a and 2b exerted the strongest antiviral activity against HEV. In conclusion, these data demonstrate for the first time moderate anti-HEV activities of types II and III IFNs and different IFN-α subtypes. As HEV employed a potent anti-interferon mechanism by restricting ISG expression, exogenous application of IFNs as immunotherapy should be carefully assessed.

scholarly journals GCD14p, a Repressor of GCN4 Translation, Cooperates with Gcd10p and Lhp1p in the Maturation of Initiator Methionyl-tRNA in Saccharomyces cerevisiae

1999 ◽  
Vol 19 (6) ◽  
pp. 4167-4181 ◽  
Author(s):  
Olga Calvo ◽  
Rafael Cuesta ◽  
James Anderson ◽  
Noelia Gutiérrez ◽  
Minerva Teresa García-Barrio ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Growth ◽  
Rna Polymerase Iii ◽  
Mrna Translation ◽  
Direct Function ◽  
Binding Motifs ◽  
La Protein ◽  
High Copy Number Plasmid ◽  
Essential Function

ABSTRACT Gcd10p and Gcd14p were first identified genetically as repressors of GCN4 mRNA translation in Saccharomyces cerevisiae. Recent findings indicate that Gcd10p and Gcd14p reside in a nuclear complex required for the presence of 1-methyladenosine in tRNAs. Here we show that Gcd14p is an essential protein with predicted binding motifs forS-adenosylmethionine, consistent with a direct function in tRNA methylation. Two different gcd14 mutants exhibit defects in cell growth and accumulate high levels of initiator methionyl-tRNA (tRNAi Met) precursors containing 5′ and 3′ extensions, suggesting a defect in processing of the primary transcript. Dosage suppressors of gcd10 mutations, encoding tRNAi Met (hcIMT1 to hcIMT4; hc indicates that the gene is carried on a high-copy-number plasmid) or a homologue of human La protein implicated in tRNA 3′-end formation (hcLHP1), also suppressed gcd14 mutations. In fact, the lethality of a GCD14 deletion was suppressed by hcIMT4, indicating that the essential function of Gcd14p is required for biogenesis of tRNAi Met. A mutation inGCD10 or deletion of LHP1 exacerbated the defects in cell growth and expression of mature tRNAi Met in gcd14 mutants, consistent with functional interactions between Gcd14p, Gcd10p, and Lhp1p in vivo. Surprisingly, the amounts of NME1 and RPR1, the RNA components of RNases P and MRP, were substantially lower in gcd14 lhp1::LEU2 double mutants than in the corresponding single mutants, whereas 5S rRNA was present at wild-type levels. Our findings suggest that Gcd14p and Lhp1p cooperate in the maturation of a subset of RNA polymerase III transcripts.

scholarly journals Twitchin kinase inhibits muscle activity

2017 ◽  
Vol 28 (12) ◽  
pp. 1591-1600 ◽  
Author(s):  
Yohei Matsunaga ◽  
Hyundoo Hwang ◽  
Barbara Franke ◽  
Rhys Williams ◽  
McKenna Penley ◽  
...  
Keyword(s):  
Muscle Activity ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Wild Type ◽  
Competitive Fitness ◽  
Binding Partners ◽  
Fibronectin Domains ◽  
First Time

Muscle sarcomeres contain giant polypeptides composed of multiple immunoglobulin and fibronectin domains and one or two protein kinase domains. Although binding partners for a number of this family’s kinase domains have been identified, the catalytic necessity of these kinase domains remains unknown. In addition, various members of this kinase family are suspected pseudokinases with no or little activity. Here we address catalytic necessity for the first time, using the prototypic invertebrate representative twitchin (UNC-22) from Caenorhabditis elegans. In in vitro experiments, change of a conserved lysine (K) that is involved in ATP coordination to alanine (A) resulted in elimination of kinase activity without affecting the overall structure of the kinase domain. The same mutation, unc-22(sf21), was generated in the endogenous twitchin gene. The unc-22(sf21) worms have well-organized sarcomeres. However, unc-22(sf21) mutants move faster than wild-type worms and, by optogenetic experiments, contract more. Wild-type nematodes exhibited greater competitive fitness than unc-22(sf21) mutants. Thus the catalytic activity of twitchin kinase has a role in vivo, where it inhibits muscle activity and is likely maintained by selection.

scholarly journals Expression and Functional Characterization of the First Bacteriophage-Encoded Chaperonin

2012 ◽  
Vol 86 (18) ◽  
pp. 10103-10111 ◽  
Author(s):  
Lidia P. Kurochkina ◽  
Pavel I. Semenyuk ◽  
Victor N. Orlov ◽  
Johan Robben ◽  
Nina N. Sykilinda ◽  
...  
Keyword(s):  
Thermal Inactivation ◽  
Functional Characterization ◽  
Content Type ◽  
Chaperonin Groel ◽  
Phage Propagation ◽  
Physicochemical Methods ◽  
First Time

Chaperonins promote protein foldingin vivoand are ubiquitously found in bacteria, archaea, and eukaryotes. The first viral chaperonin GroEL ortholog, gene product 146 (gp146), whose gene was earlier identified in the genome of bacteriophage EL, has been shown to be synthesized during phage propagation inPseudomonas aeruginosacells. The recombinant gp146 has been expressed inEscherichia coliand characterized by different physicochemical methods for the first time. Using serum against the recombinant protein, gp146's native substrate, the phage endolysin gp188, has been immunoprecipitated from the lysate of EL-infected bacteria and identified by mass spectrometry.In vitroexperiments have shown that gp146 has a protective effect against endolysin thermal inactivation and aggregation, providing evidence of its chaperonin function. The phage chaperonin has been found to have the architecture and some properties similar to those of GroEL but not to require cochaperonin for its functional activity.

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