scholarly journals Physical and Genetic Interaction between Ammonium Transporters and the Signaling Protein Rho1 in the Plant Pathogen Ustilago maydis

2014 ◽  
Vol 13 (10) ◽  
pp. 1328-1336 ◽  
Author(s):  
Jinny A. Paul ◽  
Michelle T. Barati ◽  
Michael Cooper ◽  
Michael H. Perlin
Keyword(s):  
Yellow Fluorescent Protein ◽  
Ustilago Maydis ◽  
Small Gtpase ◽  
Ammonium Transporter ◽  
Fundamental Aspect ◽  
Physical Interaction ◽  
Content Type ◽  
Signaling Protein ◽  
Ammonium Transporters

ABSTRACTDimorphic transitions between yeast-like and filamentous forms occur in many fungi and are often associated with pathogenesis. One of the cues for such a dimorphic switch is the availability of nutrients. Under conditions of nitrogen limitation, fungal cells (such as those ofSaccharomyces cerevisiaeandUstilago maydis) switch from budding to pseudohyphal or filamentous growth. Ammonium transporters (AMTs) are responsible for uptake and, in some cases, for sensing the availability of ammonium, a preferred nitrogen source. Homodimer and/or heterodimer formation may be required for regulating the activity of the AMTs. To investigate the potential interactions of Ump1 and Ump2, the AMTs of the maize pathogenU. maydis, we first used the split-ubiquitin system, followed by a modified split-YFP (yellow fluorescent protein) system, to validate the interactionsin vivo. This analysis showed the formation of homo- and hetero-oligomers by Ump1 and Ump2. We also demonstrated the interaction of the high-affinity ammonium transporter, Ump2, with the Rho1 GTPase, a central protein in signaling, with roles in controlling polarized growth. This is the first demonstration in eukaryotes of the physical interactionin vivoof an ammonium transporter with the signaling protein Rho1. Moreover, the Ump proteins interact with Rho1 during the growth of cells in low ammonium concentrations, a condition required for the expression of the Umps. Based on these results and the genetic evidence for the interaction of Ump2 with both Rho1 and Rac1, another small GTPase, we propose a model for the role of these interactions in controlling filamentation, a fundamental aspect of development and pathogenesis inU. maydis.

scholarly journals Interaction between ROCK II and Nucleophosmin/B23 in the Regulation of Centrosome Duplication

2006 ◽  
Vol 26 (23) ◽  
pp. 9016-9034 ◽  
Author(s):  
Zhiyong Ma ◽  
Masayuki Kanai ◽  
Kenji Kawamura ◽  
Kozo Kaibuchi ◽  
Keqiang Ye ◽  
...  
Keyword(s):  
Cyclin E ◽  
Active Form ◽  
Small Gtpase ◽  
Centrosome Duplication ◽  
Physical Interaction ◽  
Dependent Manner ◽  
Physical Separation ◽  
Initial Event ◽  
Activity Dependent

ABSTRACT Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr199 by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr199 remains at centrosomes. It has been shown that Thr199 phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr199. Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr199-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.

scholarly journals BEM46 Shows Eisosomal Localization and Association with Tryptophan-Derived Auxin Pathway in Neurospora crassa

2014 ◽  
Vol 13 (8) ◽  
pp. 1051-1063 ◽  
Author(s):  
K. Kollath-Leiß ◽  
C. Bönniger ◽  
P. Sardar ◽  
F. Kempken
Keyword(s):  
Neurospora Crassa ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Yellow Fluorescent Protein ◽  
Content Type ◽  
Bioinformatic Tools ◽  
Ascospore Germination ◽  
Alternatively Spliced ◽  
Indole Production

ABSTRACTBEM46 proteins are evolutionarily conserved, but their functions remain elusive. We reported previously that the BEM46 protein inNeurospora crassais targeted to the endoplasmic reticulum (ER) and is essential for ascospore germination. In the present study, we established abem46knockout strain ofN. crassa. This Δbem46mutant exhibited a level of ascospore germination lower than that of the wild type but much higher than those of the previously characterizedbem46-overexpressing and RNA interference (RNAi) lines. Reinvestigation of the RNAi transformants revealed two types of alternatively splicedbem46mRNA; expression of either type led to a loss of ascospore germination. Our results indicated that the phenotype was not due tobem46mRNA downregulation or loss but was caused by the alternatively spliced mRNAs and the peptides they encoded. Using theN. crassaortholog of the eisosomal protein PILA fromAspergillus nidulans, we further demonstrated the colocalization of BEM46 with eisosomes. Employing the yeast two-hybrid system, we identified a single interaction partner: anthranilate synthase component II (encoded bytrp-1). This interaction was confirmedin vivoby a split-YFP (yellow fluorescent protein) approach. The Δtrp-1mutant showed reduced ascospore germination and increased indole production, and we used bioinformatic tools to identify a putative auxin biosynthetic pathway. The genes involved exhibited various levels of transcriptional regulation in the differentbem46transformant and mutant strains. We also investigated the indole production of the strains in different developmental stages. Our findings suggested that the regulation of indole biosynthesis genes was influenced bybem46overexpression. Furthermore, we uncovered evidence of colocalization of BEM46 with the neutral amino acid transporter MTR.

scholarly journals Dynamics of Mitochondrial RNA-Binding Protein Complex in Trypanosoma brucei and Its Petite Mutant under Optimized Immobilization Conditions

2014 ◽  
Vol 13 (9) ◽  
pp. 1232-1240 ◽  
Author(s):  
Zhenqiu Huang ◽  
Sabine Kaltenbrunner ◽  
Eva Šimková ◽  
David Stanĕk ◽  
Julius Lukeš ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Rna Binding ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Yellow Fluorescent Protein ◽  
Rna Binding Protein ◽  
Mitochondrial Rna ◽  
Content Type ◽  
Petite Mutant

ABSTRACT There are a variety of complex metabolic processes ongoing simultaneously in the single, large mitochondrion of Trypanosoma brucei . Understanding the organellar environment and dynamics of mitochondrial proteins requires quantitative measurement in vivo . In this study, we have validated a method for immobilizing both procyclic stage (PS) and bloodstream stage (BS) T. brucei brucei with a high level of cell viability over several hours and verified its suitability for undertaking fluorescence recovery after photobleaching (FRAP), with mitochondrion-targeted yellow fluorescent protein (YFP). Next, we used this method for comparative analysis of the translational diffusion of mitochondrial RNA-binding protein 1 (MRP1) in the BS and in T. b. evansi . The latter flagellate is like petite mutant Saccharomyces cerevisiae because it lacks organelle-encoded nucleic acids. FRAP measurement of YFP-tagged MRP1 in both cell lines illuminated from a new perspective how the absence or presence of RNA affects proteins involved in mitochondrial RNA metabolism. This work represents the first attempt to examine this process in live trypanosomes.

scholarly journals Formation of Polyphosphate by Polyphosphate Kinases and Its Relationship to Poly(3-Hydroxybutyrate) Accumulation in Ralstonia eutropha Strain H16

2015 ◽  
Vol 81 (24) ◽  
pp. 8277-8293 ◽  
Author(s):  
Tony Tumlirsch ◽  
Anna Sznajder ◽  
Dieter Jendrossek
Keyword(s):  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Proteome Analysis ◽  
Enhanced Yellow Fluorescent Protein ◽  
Granule Formation ◽  
Content Type ◽  
Cell Extracts ◽  
C And N

ABSTRACTA protein (PhaX) that interacted with poly(3-hydroxybutyrate) (PHB) depolymerase PhaZa1 and with PHB granule-associated phasin protein PhaP2 was identified by two-hybrid analysis. Deletion ofphaXresulted in an increase in the level of polyphosphate (polyP) granule formation and in impairment of PHB utilization in nutrient broth-gluconate cultures. A procedure for enrichment of polyP granules from cell extracts was developed. Twenty-seven proteins that were absent in other cell fractions were identified in the polyP granule fraction by proteome analysis. One protein (A2437) harbored motifs characteristic of type 1 polyphosphate kinases (PPK1s), and two proteins (A1212, A1271) had PPK2 motifs.In vivocolocalization with polyP granules was confirmed by expression of C- and N-terminal fusions of enhanced yellow fluorescent protein (eYFP) with the three polyphosphate kinases (PPKs). Screening of the genome DNA sequence for additional proteins with PPK motifs revealed one protein with PPK1 motifs and three proteins with PPK2 motifs. Construction and subsequent expression of C- and N-terminal fusions of the four new PPK candidates with eYFP showed that only A1979 (PPK2 motif) colocalized with polyP granules. The other three proteins formed fluorescent foci near the cell pole (apart from polyP) (A0997, B1019) or were soluble (A0226). Expression of theRalstonia eutropha ppk(ppkReu) genes in anEscherichia coliΔppkbackground and construction of a set of single and multiple chromosomal deletions revealed that both A2437 (PPK1a) and A1212 (PPK2c) contributed to polyP granule formation. Mutants with deletion of both genes were unable to produce polyP granules. The formation and utilization of PHB and polyP granules were investigated in different chromosomal backgrounds.

scholarly journals Comparative Proteome Analysis Reveals Four Novel Polyhydroxybutyrate (PHB) Granule-Associated Proteins in Ralstonia eutropha H16

2014 ◽  
Vol 81 (5) ◽  
pp. 1847-1858 ◽  
Author(s):  
Anna Sznajder ◽  
Daniel Pfeiffer ◽  
Dieter Jendrossek
Keyword(s):  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
Proteome Analysis ◽  
Detectable Effect ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Associated Proteins ◽  
Phb Synthase

ABSTRACTIdentification of proteins that were present in a polyhydroxybutyrate (PHB) granule fraction isolated fromRalstonia eutrophabut absent in the soluble, membrane, and membrane-associated fractions revealed the presence of only 12 polypeptides with PHB-specific locations plus 4 previously known PHB-associated proteins with multiple locations. None of the previously postulated PHB depolymerase isoenzymes (PhaZa2 to PhaZa5, PhaZd1, and PhaZd2) and none of the two known 3-hydroxybutyrate oligomer hydrolases (PhaZb and PhaZc) were significantly present in isolated PHB granules. Four polypeptides were found that had not yet been identified in PHB granules. Three of the novel proteins are putative α/β-hydrolases, and two of those (A0671 and B1632) have a PHB synthase/depolymerase signature. The third novel protein (A0225) is a patatin-like phospholipase, a type of enzyme that has not been described for PHB granules of any PHB-accumulating species. No function has been ascribed to the fourth protein (A2001), but its encoding gene forms an operon withphaB2(acetoacetyl-coenzyme A [CoA] reductase) andphaC2(PHB synthase), and this is in line with a putative function in PHB metabolism. The localization of the four new proteins at the PHB granule surface was confirmedin vivoby fluorescence microscopy of constructed fusion proteins with enhanced yellow fluorescent protein (eYFP). Deletion of A0671 and B1632 had a minor but detectable effect on the PHB mobilization ability in the stationary growth phase of nutrient broth (NB)-gluconate cells, confirming the functional involvement of both proteins in PHB metabolism.

The small GTPase Rac1 is a novel binding partner of Bcl-2 and stabilizes its antiapoptotic activity

Blood ◽  
2011 ◽  
Vol 117 (23) ◽  
pp. 6214-6226 ◽  
Author(s):  
Rathiga Velaithan ◽  
Jia Kang ◽  
Jayshree L. Hirpara ◽  
Thomas Loh ◽  
Boon Cher Goh ◽  
...  
Keyword(s):  
B Cell Lymphoma ◽  
Small Gtpase ◽  
Superoxide Production ◽  
Leukemia Cells ◽  
Physical Interaction ◽  
Human Leukemia ◽  
Noncancerous Tissue ◽  
Functional Inhibition

Abstract The small GTPase Rac1 is involved in the activation of the reduced NAD phosphate oxidase complex resulting in superoxide production. We recently showed that Bcl-2 overexpression inhibited apoptosis in leukemia cells by creating a pro-oxidant intracellular milieu, and that inhibiting intracellular superoxide production sensitized Bcl-2–overexpressing cells to apoptotic stimuli. We report here that silencing and functional inhibition of Rac1 block Bcl-2–mediated increase in intracellular superoxide levels in tumor cells. Using confocal, electron microscopy and coimmunoprecipitation, as well as glutathione S-transferase–fusion proteins, we provide evidence for a colocalization and physical interaction between the 2 proteins. This interaction is blocked in vitro and in vivo by the BH3 mimetics as well as by synthetic Bcl-2 BH3 domain peptides. That this interaction is functionally relevant is supported by the ability of the Bcl-2 BH3 peptide as well as the silencing and functional inhibition of Rac1 to inhibit intracellular superoxide production as well as overcome Bcl-2–mediated drug resistance in human leukemia cells and cervical cancer cells. Notably, the interaction was observed in primary cells derived from patients with B-cell lymphoma overexpressing Bcl-2 but not in noncancerous tissue. These data provide a novel facet in the biology of Bcl-2 with potential implications for targeted anticancer drug design.

scholarly journals Functional Analysis of the Single Est1/Ebs1 Homologue in Kluyveromyces lactis Reveals Roles in both Telomere Maintenance and Rapamycin Resistance

2012 ◽  
Vol 11 (7) ◽  
pp. 932-942 ◽  
Author(s):  
Min Hsu ◽  
Eun Young Yu ◽  
Ondrej Sprušanský ◽  
Michael J. McEachern ◽  
Neal F. Lue
Keyword(s):  
Functional Analysis ◽  
Budding Yeast ◽  
Kluyveromyces Lactis ◽  
Telomere Maintenance ◽  
Physical Interaction ◽  
Content Type ◽  
Terminal Domain ◽  
Cell Extracts

ABSTRACT Est1 and Ebs1 in Saccharomyces cerevisiae are paralogous proteins that arose through whole-genome duplication and that serve distinct functions in telomere maintenance and translational regulation. Here we present our functional analysis of the sole Est1/Ebs1 homologue in the related budding yeast Kluyveromyces lactis (named Kl Est1). We show that similar to other Est1s, Kl Est1 is required for normal telomere maintenance in vivo and full telomerase primer extension activity in vitro . Kl Est1 also associates with telomerase RNA (Ter1) and an active telomerase complex in cell extracts. Both the telomere maintenance and the Ter1 association functions of Kl Est1 require its N-terminal domain but not its C terminus. Analysis of clusters of point mutations revealed residues in both the N-terminal TPR subdomain and the downstream helical subdomain (DSH) that are important for telomere maintenance and Ter1 association. A UV cross-linking assay was used to establish a direct physical interaction between Kl Est1 and a putative stem-loop in Ter1, which also requires both the TPR and DSH subdomains. Moreover, similar to S. cerevisiae Ebs1 ( Sc Ebs1) (but not Sc Est1), Kl Est1 confers rapamycin sensitivity and may be involved in nonsense-mediated decay. Interestingly, unlike telomere regulation, this apparently separate function of Kl Est1 requires its C-terminal domain. Our findings provide insights on the mechanisms and evolution of Est1/Ebs1 homologues in budding yeast and present an attractive model system for analyzing members of this multifunctional protein family.

scholarly journals To Be or Not To Be a Poly(3-Hydroxybutyrate) (PHB) Depolymerase: PhaZd1 (PhaZ6) and PhaZd2 (PhaZ7) of Ralstonia eutropha, Highly Active PHB Depolymerases with No Detectable Role in Mobilization of Accumulated PHB

2014 ◽  
Vol 80 (16) ◽  
pp. 4936-4946 ◽  
Author(s):  
Anna Sznajder ◽  
Dieter Jendrossek
Keyword(s):  
Fluorescent Protein ◽  
Ralstonia Eutropha ◽  
Yellow Fluorescent Protein ◽  
High Capacity ◽  
Constitutive Expression ◽  
Enhanced Yellow Fluorescent Protein ◽  
Content Type ◽  
Phb Depolymerase

ABSTRACTThe putative physiological functions of two related intracellular poly(3-hydroxybutyrate) (PHB) depolymerases, PhaZd1 and PhaZd2, ofRalstonia eutrophaH16 were investigated. Purified PhaZd1 and PhaZd2 were active with native PHB granulesin vitro. Partial removal of the proteinaceous surface layer of native PHB granules by trypsin treatment or the use of PHB granules isolated from ΔphaP1or ΔphaP1-phaP5mutant strains resulted in increased specific PHB depolymerase activity, especially for PhaZd2. Constitutive expression of PhaZd1 or PhaZd2 reduced or even prevented the accumulation of PHB under PHB-permissive conditionsin vivo. Expression of translational fusions of enhanced yellow fluorescent protein (EYFP) with PhaZd1 and PhaZd2 in which the active-site serines (S190 and Ser193) were replaced with alanine resulted in the colocalization of only PhaZd1 fusions with PHB granules. C-terminal fusions of inactive PhaZd2(S193A) with EYFP revealed the presence of spindle-like structures, and no colocalization with PHB granules was observed. Chromosomal deletion ofphaZd1,phaZd2, or both depolymerase genes had no significant effect on PHB accumulation and mobilization during growth in nutrient broth (NB) or NB-gluconate medium. Moreover, neither proteome analysis of purified native PHB granules norlacZfusion studies gave any indication that PhaZd1 or PhaZd2 was detectably present in the PHB granule fraction or expressed at all during growth on NB-gluconate medium. In conclusion, PhaZd1 and PhaZd2 are two PHB depolymerases with a high capacity to degrade PHB when artificially expressed but are apparently not involved in PHB mobilization in the wild type. The truein vivofunctions of PhaZd1 and PhaZd2 remain obscure.

scholarly journals Purification of the Escherichia coli ammonium transporter AmtB reveals a trimeric stoichiometry

2002 ◽  
Vol 364 (2) ◽  
pp. 527-535 ◽  
Author(s):  
Dan BLAKEY ◽  
Andrew LEECH ◽  
Gavin H. THOMAS ◽  
Graham COUTTS ◽  
Kim FINDLAY ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Analytical Ultracentrifugation ◽  
Quaternary Structure ◽  
Gel Filtration ◽  
Ammonium Transporter ◽  
Ammonium Transporters ◽  
Sds Page ◽  
The Family ◽  
And Function

The Amt family of high-affinity ammonium transporters is a family of integral membrane proteins that are found in archaea, bacteria, fungi, plants and animals. Furthermore, the family has recently been extended to humans with the recognition that both the erythroid and non-erythroid Rhesus proteins are also ammonium transporters. The Escherichia coli AmtB protein offers a good model system for the Amt family and in order to address questions relating to both its structure and function we have overproduced a histidine-tagged form of the protein (AmtB6H) and purified it to homogeneity. We examined the quaternary structure of AmtB6H (which is active in vivo) by SDS/PAGE, gel-filtration chromatography, dynamic light scattering and sedimentation ultracentrifugation. The protein was resistant to dissociation by SDS and behaved as a stable oligomer on SDS/PAGE. By equilibrium desorption chromatography we determined the mass ratio of dodecyl β-d-maltoside to AmtB in the detergent-solubilized complex to be 1.03±0.03, and this allowed us to calculate, from analytical-ultracentrifugation data, that AmtB purifies as a trimer.

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