Long-range cooperative binding of kinesin to a microtubule in the presence of ATP

Interaction of kinesin-coated latex beads with a single microtubule (MT) was directly observed by fluorescence microscopy. In the presence of ATP, binding of a kinesin bead to the MT facilitated the subsequent binding of other kinesin beads to an adjacent region on the MT that extended for micrometers in length. This cooperative binding was not observed in the presence of ADP or 5′-adenylylimidodiphosphate (AMP-PNP), where binding along the MT was random. Cooperative binding also was induced by an engineered, heterodimeric kinesin, WT/E236A, that could hydrolyze ATP, yet remained fixed on the MT in the presence of ATP. Relative to the stationary WT/E236A kinesin on a MT, wild-type kinesin bound preferentially in close proximity, but was biased to the plus-end direction. These results suggest that kinesin binding and ATP hydrolysis may cause a long-range state transition in the MT, increasing its affinity for kinesin toward its plus end. Thus, our study highlights the active involvement of MTs in kinesin motility.

Download Full-text

The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽

10.1093/genetics/155.3.1105 ◽

2000 ◽

Vol 155 (3) ◽

pp. 1105-1117 ◽

Cited By ~ 4

Author(s):

W John Haynes ◽

Kit-Yin Ling ◽

Robin R Preston ◽

Yoshiro Saimi ◽

Ching Kung

Keyword(s):

Genomic Library ◽

Open Reading Frame ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Rt Pcr ◽

Reading Frame ◽

Close Proximity ◽

In The Wild ◽

Dna Fragment ◽

Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

Download Full-text

ParB deficiency in Pseudomonas aeruginosa destabilizes the partner protein ParA and affects a variety of physiological parameters

Microbiology ◽

10.1099/mic.0.024661-0 ◽

2009 ◽

Vol 155 (4) ◽

pp. 1080-1092 ◽

Cited By ~ 28

Author(s):

A. A. Bartosik ◽

J. Mierzejewska ◽

C. M. Thomas ◽

G. Jagura-Burdzy

Keyword(s):

Pseudomonas Aeruginosa ◽

Fluorescence Microscopy ◽

Bacterial Growth ◽

Complete Loss ◽

Physiological Parameters ◽

Wild Type ◽

Partial Removal ◽

Partner Protein ◽

The One ◽

Mutant Phenotypes

Deletions leading to complete or partial removal of ParB were introduced into the Pseudomonas aeruginosa chromosome. Fluorescence microscopy of fixed cells showed that ParB mutants lacking the C-terminal domain or HTH motif formed multiple, less intense foci scattered irregularly, in contrast to the one to four ParB foci per cell symmetrically distributed in wild-type P. aeruginosa. All parB mutations affected both bacterial growth and swarming and swimming motilities, and increased the production of anucleate cells. Similar effects were observed after inactivation of parA of P. aeruginosa. As complete loss of ParA destabilized its partner ParB it was unclear deficiency of which protein is responsible for the mutant phenotypes. Analysis of four parB mutants showed that complete loss of ParB destabilized ParA whereas three mutants that retained the N-terminal 90 aa of ParB did not. As all four parB mutants demonstrate the same defects it can be concluded that either ParB, or ParA and ParB in combination, plays an important role in nucleoid distribution, growth and motility in P. aeruginosa.

Download Full-text

Extracellular Glycanases of Rhizobium leguminosarum Are Activated on the Cell Surface by an Exopolysaccharide-Related Component

Journal of Bacteriology ◽

10.1128/jb.182.5.1304-1312.2000 ◽

2000 ◽

Vol 182 (5) ◽

pp. 1304-1312 ◽

Cited By ~ 27

Author(s):

Angeles Zorreguieta ◽

Christine Finnie ◽

J. Allan Downie

Keyword(s):

Cell Wall ◽

Agrobacterium Tumefaciens ◽

Cell Surface ◽

Rhizobium Leguminosarum ◽

Plant Cell Wall ◽

Agar Medium ◽

Wild Type ◽

Close Proximity ◽

Mutant Strains ◽

Colony Surface

ABSTRACT Rhizobium leguminosarum secretes two extracellular glycanases, PlyA and PlyB, that can degrade exopolysaccharide (EPS) and carboxymethyl cellulose (CMC), which is used as a model substrate of plant cell wall cellulose polymers. When grown on agar medium, CMC degradation occurred only directly below colonies of R. leguminosarum, suggesting that the enzymes remain attached to the bacteria. Unexpectedly, when a PlyA-PlyB-secreting colony was grown in close proximity to mutants unable to produce or secrete PlyA and PlyB, CMC degradation occurred below that part of the mutant colonies closest to the wild type. There was no CMC degradation in the region between the colonies. By growing PlyB-secreting colonies on a lawn of CMC-nondegrading mutants, we could observe a halo of CMC degradation around the colony. Using various mutant strains, we demonstrate that PlyB diffuses beyond the edge of the colony but does not degrade CMC unless it is in contact with the appropriate colony surface. PlyA appears to remain attached to the cells since no such diffusion of PlyA activity was observed. EPS defective mutants could secrete both PlyA and PlyB, but these enzymes were inactive unless they came into contact with an EPS+ strain, indicating that EPS is required for activation of PlyA and PlyB. However, we were unable to activate CMC degradation with a crude EPS fraction, indicating that activation of CMC degradation may require an intermediate in EPS biosynthesis. Transfer of PlyB to Agrobacterium tumefaciens enabled it to degrade CMC, but this was only observed if it was grown on a lawn ofR. leguminosarum. This indicates that the surface ofA. tumefaciens is inappropriate to activate CMC degradation by PlyB. Analysis of CMC degradation by other rhizobia suggests that activation of secreted glycanases by surface components may occur in other species.

Download Full-text

CRISPR/Cas9-Mediated Gene Editing of Vacuolar ATPase Subunit D Mediates Phytohormone Biosynthesis and Virus Resistance in Rice

10.21203/rs.3.rs-1200905/v1 ◽

2021 ◽

Author(s):

Qinghua Lu ◽

Xiangwen Luo ◽

Xiao Yang ◽

Tong Zhou ◽

Yu Zhang ◽

...

Keyword(s):

Virus Resistance ◽

Atp Hydrolysis ◽

Proton Translocation ◽

Resistance Breeding ◽

Rice Stripe Virus ◽

Proton Pumps ◽

Wild Type ◽

Knockout Mutant ◽

Atpase Subunit ◽

Vacuolar Atpases

Abstract Background: Vacuolar ATPases (v-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis; Previous research revealed Osv-ATPases mediates phytohormes levels and resistance in rice. Osv-ATPase subunit d (Osv-ATPase d) is part of an integral, membrane-embedded V0 complex of V-ATPases complex, whether Osv-ATPase d involves in phytohormes biosynthesis and resistance in rice remains unknown.Finding: The knockout mutant line (line 5) of Osv-ATPase d was generated using the CRISPR/Cas9 system, mutation of Osv-ATPase d did not show any detrimental effect on plant growth or yield productivity. Transcriptomic results showed Osv-ATPase d probably involved in mediating the biosynthesis of plant hormones and resistance in rice. Mutation of Osv-ATPase d significantly increased JA and ABA biosynthesis than wild type. Compared to wild type, mutation of Osv-ATPase d increased the resistance against Southern rice black-streaked dwarf virus (SRBSDV), however, decreased the resistance against Rice stripe virus (RSV) in rice. Conclusion: Taken together, our data reveal the Osv-ATPase d mediates phytohormone biosynthesis and virus resistance in rice, which can be selected as a potential target for resistance breeding in rice.

Download Full-text

UBL domain of Usp14 and other proteins stimulates proteasome activities and protein degradation in cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1808731115 ◽

2018 ◽

Vol 115 (50) ◽

pp. E11642-E11650 ◽

Cited By ~ 19

Author(s):

Hyoung Tae Kim ◽

Alfred L. Goldberg

Keyword(s):

Protein Degradation ◽

Atp Hydrolysis ◽

Deubiquitinating Enzyme ◽

Wild Type ◽

Peptide Hydrolysis ◽

Ubiquitin Chain ◽

Important Regulator ◽

General Response ◽

Ubiquitin Conjugate ◽

Stimulation Of

The best-known function of ubiquitin-like (UBL) domains in proteins is to enable their binding to 26S proteasomes. The proteasome-associated deubiquitinating enzyme Usp14/UBP6 contains an N-terminal UBL domain and is an important regulator of proteasomal activity. Usp14 by itself represses multiple proteasomal activities but, upon binding a ubiquitin chain, Usp14 stimulates these activities and promotes ubiquitin-conjugate degradation. Here, we demonstrate that Usp14’s UBL domain alone mimics this activation of proteasomes by ubiquitin chains. Addition of this UBL domain to purified 26S proteasomes stimulated the same activities inhibited by Usp14: peptide entry and hydrolysis, protein-dependent ATP hydrolysis, deubiquitination by Rpn11, and the degradation of ubiquitinated and nonubiquitinated proteins. Thus, the binding of Usp14’s UBL (apparently to Rpn1’s T2 site) seems to mediate the activation of proteasomes by ubiquitinated substrates. However, the stimulation of these various activities was greater in proteasomes lacking Usp14 than in wild-type particles and thus is a general response that does not involve some displacement of Usp14. Furthermore, the UBL domains from hHR23 and hPLIC1/UBQLN1 also stimulated peptide hydrolysis, and the expression of hHR23A’s UBL domain in HeLa cells stimulated overall protein degradation. Therefore, many UBL-containing proteins that bind to proteasomes may also enhance allosterically its proteolytic activity.

Download Full-text

Rad51 catalytic mutants differentially affect the Rad51 nucleoprotein filament in vivo

10.1101/070276 ◽

2016 ◽

Author(s):

Maureen M. Mundia ◽

Alissa C. Magwood ◽

Mark D. Baker

Keyword(s):

Homologous Recombination ◽

Dna Synthesis ◽

Cell Lines ◽

Atp Hydrolysis ◽

Dominant Negative ◽

Strand Exchange ◽

Wild Type ◽

Hybridoma Cell Lines ◽

Insight Into

ABSTRACTIn this study, we utilized mouse hybridoma cell lines stably expressing ectopic wild-type Rad51, or the Rad51-K133A and Rad51-K133R catalytic mutants deficient in ATP binding and ATP hydrolysis, respectively, to investigate effects on the Rad51 nucleoprotein filament in vivo. Immunoprecipitation studies reveal interactions between ectopic wild-type Rad51, Rad51-K133A and Rad51-K133R and endogenous Rad51, Brca2 and p53 proteins. Importantly, the expression of Rad51-K133A and Rad51-K133R catalytic mutants (but not wild-type Rad51) targets endogenous Rad51, Brca2 and p53 proteins for proteasome-mediated degradation. Expression of Rad51-K133R significantly reduces nascent DNA synthesis (3’ polymerization) during homologous recombination (HR), but the effects of Rad51-K133A on 3’ polymerization are considerably more severe. Provision of additional wild-type Rad51 in cell lines expressing Rad51-K133A or Rad51-K133R does not restore diminished levels of endogenous Brca2, Rad51 or p53, nor restore the deficiency in 3’ polymerization. Cells expressing Rad51-K133A are also significantly reduced in their capacity to drive strand exchange through regions of heterology. Our results reveal an interesting mechanistic dichotomy in the way mutant Rad51-K133A and Rad51-K133R proteins influence 3’ polymerization and provide novel insight into the mechanism of their dominant-negative phenotypes.

Download Full-text

Ensovibep, a novel trispecific DARPin candidate that protects against SARS-CoV-2 variants

10.21203/rs.3.rs-1170399/v1 ◽

2021 ◽

Author(s):

Michael Stumpp

Keyword(s):

Single Agent ◽

Standard Of Care ◽

Cooperative Binding ◽

Wild Type ◽

Inhibitory Potency ◽

Alpha Variant ◽

The Individual ◽

Very High ◽

Comprehensive Study

Abstract SARS-CoV-2 has infected millions of people globally and continues to undergo evolution. Emerging variants can be partially resistant to vaccine induced and therapeutic antibodies, emphasizing the urgent need for accessible, broad-spectrum therapeutics. Here, we report a comprehensive study of ensovibep, the first trispecific clinical DARPin candidate, that can simultaneously engage all three units of the spike protein trimer to potently inhibit ACE2 interaction, as revealed by structural analyses. The cooperative binding of the individual modules enables ensovibep to retain inhibitory potency against all frequent SARS-CoV-2 variants, including Omicron, as of December 2021. Moreover, viral passaging experiments show that ensovibep, when used as a single agent, can prevent development of escape mutations comparably to a cocktail of monoclonal antibodies (mAb). Finally, we demonstrate that the very high in vitro antiviral potency also translates into significant therapeutic protection and reduction of pathogenesis in Roborovski dwarf hamsters infected with either the SARS-CoV-2 wild-type or the Alpha variant. In this model, ensovibep prevents fatality and provides substantial protection equivalent to the standard of care mAb cocktail. These results support further clinical evaluation and indicate that ensovibep could be a valuable alternative to mAb cocktails and other treatments for COVID-19.

Download Full-text

Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

Applied and Environmental Microbiology ◽

10.1128/aem.71.2.621-628.2005 ◽

2005 ◽

Vol 71 (2) ◽

pp. 621-628 ◽

Cited By ~ 37

Author(s):

Zhi-Wei Chen ◽

Cheng-Ying Jiang ◽

Qunxin She ◽

Shuang-Jiang Liu ◽

Pei-Jin Zhou

Keyword(s):

Cytoplasmic Membrane ◽

Sulfur Oxidation ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Wild Type ◽

Immune Electron Microscopy ◽

Mutant Proteins ◽

Close Proximity ◽

Catalytic Sites

ABSTRACT Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C31 and C101-X-X-C104, in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.

Download Full-text

An Innovative Root Inoculation Method to StudyRalstonia solanacearumPathogenicity in Tomato Seedlings

Phytopathology ◽

10.1094/phyto-08-17-0291-r ◽

2018 ◽

Vol 108 (4) ◽

pp. 436-442 ◽

Cited By ~ 17

Author(s):

N. Singh ◽

T. Phukan ◽

P. L. Sharma ◽

K. Kabyashree ◽

A. Barman ◽

...

Keyword(s):

Fluorescence Microscopy ◽

Ralstonia Solanacearum ◽

Virulence Genes ◽

Wild Type ◽

Gus Staining ◽

Tomato Seedlings ◽

Inoculation Method ◽

Virulence Assay ◽

Bacterial Inoculum ◽

Adult Plants

In this study, we report Ralstonia solanacearum pathogenicity in the early stages of tomato seedlings by an innovative root inoculation method. Pathogenicity assays were performed under gnotobiotic conditions in microfuge tubes by employing only 6- to 7-day-old tomato seedlings for root inoculation. Tomato seedlings inoculated by this method exhibited the wilted symptom within 48 h and the virulence assay can be completed in 2 weeks. Colonization of the wilted seedlings by R. solanacearum was confirmed by using gus staining as well as fluorescence microscopy. Using this method, mutants in different virulence genes such as hrpB, phcA, and pilT could be clearly distinguished from wild-type R. solanacearum. The method described here is economic in terms of space, labor, and cost as well as the required quantity of bacterial inoculum. Thus, the newly developed assay is an easy and useful approach for investigating virulence functions of the pathogen at the seedling stage of hosts, and infection under these conditions appears to require pathogenicity mechanisms used by the pathogen for infection of adult plants.

Download Full-text

Murine Leukemia Virus Particle Assembly Quantitated by Fluorescence Microscopy: Role of Gag-Gag Interactions and Membrane Association

Journal of Virology ◽

10.1128/jvi.77.21.11651-11660.2003 ◽

2003 ◽

Vol 77 (21) ◽

pp. 11651-11660 ◽

Cited By ~ 27

Author(s):

Mariam Andrawiss ◽

Yasuhiro Takeuchi ◽

Lindsay Hewlett ◽

Mary Collins

Keyword(s):

Fluorescence Microscopy ◽

Murine Leukemia Virus ◽

Fluorescent Proteins ◽

Leukemia Virus ◽

Particle Formation ◽

Membrane Association ◽

Wild Type ◽

Particle Assembly ◽

Gag Assembly ◽

Murine Leukemia

ABSTRACT In order to track the assembly of murine leukemia virus (MLV), we used fluorescence microscopy to visualize particles containing Gag molecules fused to fluorescent proteins (FPs). Gag-FP chimeras budded from cells to produce fluorescent spots, which passed through the same pore-size filters and sedimented at the same velocity as authentic MLV. N-terminal myristylation of Gag-FPs was necessary for particle formation unless wild-type Gag was coexpressed. By labeling nonmyristylated Gag with yellow FP and wild-type Gag with cyan FP, we could quantitate the coincorporation of two proteins into single particles. This experiment showed that nonmyristylated Gag was incorporated into mixed particles at approximately 50% the efficiency of wild-type Gag. Mutations that inhibit Gag-Gag interactions (K. Alin and S. P. Goff, Virology 216:418-424, 1996; K. Alin and S. P. Goff, Virology 222:339-351, 1996) were then introduced into the capsid (CA) region of Gag-FPs. The mutations P150L and R119C/P133L inhibited fluorescent particle formation by these Gag-FPs, but Gag-FPs containing these mutations could be efficiently incorporated into particles when coexpressed with wild-type Gag. When these mutations were introduced into nonmyristylated Gag-FPs, no incorporation into particles in the presence of wild-type Gag was detected. These data suggest that two independent mechanisms, CA interactions and membrane association following myristylation, cooperate in MLV Gag assembly and budding.

Download Full-text