Reversible ADP-Ribosylation of Dinitrogenase Reductase from Rhodospirillum rubrum Regulates the Activity of the Enzyme In Vivo and In Vitro

1989 ◽  
pp. 18-25
Author(s):  
Paul W. Ludden ◽  
Scott A. Murrell ◽  
Robert G. Lowery ◽  
Wayne P. Fitzmaurice ◽  
Mark R. Pope ◽  
...  
Keyword(s):  
Rhodospirillum Rubrum ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase

scholarly journals Role of the Dinitrogenase Reductase Arginine 101 Residue in Dinitrogenase Reductase ADP-Ribosyltransferase Binding, NAD Binding, and Cleavage

2001 ◽  
Vol 183 (1) ◽  
pp. 250-256 ◽  
Author(s):  
Yan Ma ◽  
Paul W. Ludden
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT Dinitrogenase reductase is posttranslationally regulated by dinitrogenase reductase ADP-ribosyltransferase (DRAT) via ADP-ribosylation of the arginine 101 residue in some bacteria.Rhodospirillum rubrum strains in which the arginine 101 of dinitrogenase reductase was replaced by tyrosine, phenylalanine, or leucine were constructed by site-directed mutagenesis of thenifH gene. The strain containing the R101F form of dinitrogenase reductase retains 91%, the strain containing the R101Y form retains 72%, and the strain containing the R101L form retains only 28% of in vivo nitrogenase activity of the strain containing the dinitrogenase reductase with arginine at position 101. In vivo acetylene reduction assays, immunoblotting with anti-dinitrogenase reductase antibody, and [adenylate-32P]NAD labeling experiments showed that no switch-off of nitrogenase activity occurred in any of the three mutants and no ADP-ribosylation of altered dinitrogenase reductases occurred either in vivo or in vitro. Altered dinitrogenase reductases from strains UR629 (R101Y) and UR630 (R101F) were purified to homogeneity. The R101F and R101Y forms of dinitrogenase reductase were able to form a complex with DRAT that could be chemically cross-linked by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The R101F form of dinitrogenase reductase and DRAT together were not able to cleave NAD. This suggests that arginine 101 is not critical for the binding of DRAT to dinitrogenase reductase but that the availability of arginine 101 is important for NAD cleavage. Both DRAT and dinitrogenase reductase can be labeled by [carbonyl-14C]NAD individually upon UV irradiation, but most 14C label is incorporated into DRAT when both proteins are present. The ability of R101F dinitrogenase reductase to be labeled by [carbonyl-14C]NAD suggested that Arg 101 is not absolutely required for NAD binding.

Regulation of nitrogenase in the photosynthetic bacteriumRhodobacter sphaeroidescontainingdraTGandnifHDKgenes fromRhodobacter capsulatus

2001 ◽  
Vol 47 (3) ◽  
pp. 206-212 ◽  
Author(s):  
Alexander F Yakunin ◽  
Alexander S Fedorov ◽  
Tatyana V Laurinavichene ◽  
Vadim M Glaser ◽  
Nikolay S Egorov ◽  
...  
Keyword(s):  
Photosynthetic Bacteria ◽  
Rhodospirillum Rubrum ◽  
Rhodobacter Capsulatus ◽  
Nitrogenase Activity ◽  
Photosynthetic Bacterium ◽  
Adp Ribosylation ◽  
Fe Protein ◽  
Dinitrogenase Reductase ◽  
Different Strains

The photosynthetic bacteria Rhodobacter capsulatus and Rhodospirillum rubrum regulate their nitrogenase activity by the reversible ADP-ribosylation of nitrogenase Fe-protein in response to ammonium addition or darkness. This regulation is mediated by two enzymes, dinitrogenase reductase ADP-ribosyl transferase (DRAT) and dinitrogenase reductase activating glycohydrolase (DRAG). Recently, we demonstrated that another photosynthetic bacterium, Rhodobacter sphaeroides, appears to have no draTG genes, and no evidence of Fe-protein ADP-ribosylation was found in this bacterium under a variety of growth and incubation conditions. Here we show that four different strains of Rba. sphaeroides are incapable of modifying Fe-protein, whereas four out of five Rba. capsulatus strains possess this ability. Introduction of Rba. capsulatus draTG and nifHDK (structural genes for nitrogenase proteins) into Rba. sphaeroides had no effect on in vivo nitrogenase activity and on nitrogenase switch-off by ammonium. However, transfer of draTG from Rba. capsulatus was sufficient to confer on Rba. sphaeroides the ability to reversibly modify the nitrogenase Fe-protein in response to either ammonium addition or darkness. These data suggest that Rba. sphaeroides, which lacks DRAT and DRAG, possesses all the elements necessary for the transduction of signals generated by ammonium or darkness to these proteins.Key words: nitrogenase regulation, nitrogenase modification, photosynthetic bacteria.

scholarly journals Effects of Perturbations of the Nitrogenase Electron Transfer Chain on Reversible ADP-Ribosylation of Nitrogenase Fe Protein in Klebsiella pneumoniae Strains Bearing the Rhodospirillum rubrum dra Operon

2000 ◽  
Vol 182 (13) ◽  
pp. 3681-3687 ◽  
Author(s):  
Cale M. Halbleib ◽  
Yaoping Zhang ◽  
Gary P. Roberts ◽  
Paul W. Ludden
Keyword(s):  
Klebsiella Pneumoniae ◽  
Affect Regulation ◽  
Redox State ◽  
Rhodospirillum Rubrum ◽  
Reversible Inactivation ◽  
Adp Ribosylation ◽  
Fe Protein ◽  
Energy Limitation ◽  
Dinitrogenase Reductase

ABSTRACT The redox state of nitrogenase Fe protein is shown to affect regulation of ADP-ribosylation in Klebsiella pneumoniaestrains transformed by plasmids carrying dra genes fromRhodospirillum rubrum. The dra operon encodes dinitrogenase reductase ADP-ribosyltransferase and dinitrogenase reductase-activating glycohydrolase, enzymes responsible for the reversible inactivation, via ADP-ribosylation, of nitrogenase Fe protein in R. rubrum. In bacteria containing thedra operon in their chromosomes, inactivation occurs in response to energy limitation or nitrogen sufficiency. Thedra gene products, expressed at a low level in K. pneumoniae, enable transformants to reversibly ADP-ribosylate nitrogenase Fe protein in response to the presence of fixed nitrogen. The activities of both regulatory enzymes are regulated in vivo as described in R. rubrum. Genetic perturbations of the nitrogenase electron transport chain were found to affect the rate of inactivation of Fe protein. Strains lacking the electron donors to Fe protein (NifF or NifJ) were found to inactivate Fe protein more quickly than a strain with wild-type background. Deletion ofnifD, which encodes a subunit of nitrogenase MoFe protein, was found to result in a slower inactivation response. No variation was found in the reactivation responses of these strains. It is concluded that the redox state of the Fe protein contributes to the regulation of the ADP-ribosylation of Fe protein.

scholarly journals ADP-ribosylation of integrin α7 modulates the binding of integrin α7β1 to laminin

2004 ◽  
Vol 385 (1) ◽  
pp. 309-317 ◽  
Author(s):  
Zhefeng ZHAO ◽  
Joanna GRUSZCZYNSKA-BIEGALA ◽  
Anna ZOLKIEWSKA
Keyword(s):  
C2c12 Myotubes ◽  
Post Translational Modification ◽  
Integrin Β1 ◽  
Adp Ribosylation ◽  
In Vitro Binding ◽  
Vitro Binding ◽  
C2c12 Skeletal Muscle Cells ◽  
Adp Ribosyltransferase

The extracellular domain of integrin α7 is ADP-ribosylated by an arginine-specific ecto-ADP-ribosyltransferase after adding exogenous NAD+ to intact C2C12 skeletal muscle cells. The effect of ADP-ribosylation on the structure or function of integrin α7β1 has not been explored. In the present study, we show that ADP-ribosylation of integrin α7 takes place exclusively in differentiated myotubes and that this post-translational modification modulates the affinity of α7β1 dimer for its ligand, laminin. ADP-ribosylation in the 37-kDa ‘stalk’ region of α7 that takes place at micromolar NAD+ concentrations increases the binding of the α7β1 dimer to laminin. Increased in vitro binding of integrin α7β1 to laminin after ADP-ribosylation of the 37-kDa fragment of α7 requires the presence of Mn2+ and it is not observed in the presence of Mg2+. In contrast, ADP-ribosylation of the 63-kDa N-terminal region comprising the ligand-binding site of α7 that occurs at approx. 100 μM NAD+ inhibits the binding of integrin α7β1 to laminin. Furthermore, incubation of C2C12 myotubes with NAD+ increases the expression of an epitope on integrin β1 subunit recognized by monoclonal antibody 9EG7. We discuss our results based on the current models of integrin activation. We also hypothesize that ADP-ribosylation may represent a mechanism of regulation of integrin α7β1 function in myofibres in vivo when the continuity of the membrane is compromised and NAD+ is available as a substrate for ecto-ADP-ribosylation.

scholarly journals Adp Ribosylation Factor-like Protein 2 (Arl2) Regulates the Interaction of Tubulin-Folding Cofactor D with Native Tubulin

2000 ◽  
Vol 149 (5) ◽  
pp. 1087-1096 ◽  
Author(s):  
Arunashree Bhamidipati ◽  
Sally A. Lewis ◽  
Nicholas J. Cowan
Keyword(s):  
Cultured Cells ◽  
Gtpase Activating Protein ◽  
Adp Ribosylation ◽  
Chaperone Protein ◽  
Ras Family ◽  
Mutant Forms ◽  
Adp Ribosylation Factor ◽  
Β Tubulin

The ADP ribosylation factor-like proteins (Arls) are a family of small monomeric G proteins of unknown function. Here, we show that Arl2 interacts with the tubulin-specific chaperone protein known as cofactor D. Cofactors C, D, and E assemble the α/β- tubulin heterodimer and also interact with native tubulin, stimulating it to hydrolyze GTP and thus acting together as a β-tubulin GTPase activating protein (GAP). We find that Arl2 downregulates the tubulin GAP activity of C, D, and E, and inhibits the binding of D to native tubulin in vitro. We also find that overexpression of cofactors D or E in cultured cells results in the destruction of the tubulin heterodimer and of microtubules. Arl2 specifically prevents destruction of tubulin and microtubules by cofactor D, but not by cofactor E. We generated mutant forms of Arl2 based on the known properties of classical Ras-family mutations. Experiments using these altered forms of Arl2 in vitro and in vivo demonstrate that it is GDP-bound Arl2 that interacts with cofactor D, thereby averting tubulin and microtubule destruction. These data establish a role for Arl2 in modulating the interaction of tubulin-folding cofactors with native tubulin in vivo.

scholarly journals ADP Ribosylation Factor 1 Is Required for Synaptic Vesicle Budding in PC12 Cells

1997 ◽  
Vol 138 (3) ◽  
pp. 505-515 ◽  
Author(s):  
Victor Faúndez ◽  
Jim-Tong Horng ◽  
Regis B. Kelly
Keyword(s):  
Pc12 Cell ◽  
Cell Membranes ◽  
Brefeldin A ◽  
T Antigen ◽  
Gtpase Activity ◽  
Vesicle Budding ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Carrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein–T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041–1049). A peptide spanning the effector domain of human ARF1 (2–17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

scholarly journals Correlation of Activity Regulation and Substrate Recognition of the ADP-Ribosyltransferase That Regulates Nitrogenase Activity in Rhodospirillum rubrum

1999 ◽  
Vol 181 (5) ◽  
pp. 1698-1702 ◽  
Author(s):  
Kitai Kim ◽  
Yaoping Zhang ◽  
Gary P. Roberts
Keyword(s):  
Rhodospirillum Rubrum ◽  
Nitrogenase Activity ◽  
Substrate Recognition ◽  
Posttranslational Regulation ◽  
Activity Regulation ◽  
Adp Ribosylation ◽  
Dinitrogenase Reductase ◽  
Adp Ribosyltransferase

ABSTRACT In Rhodospirillum rubrum, nitrogenase activity is regulated posttranslationally through the ADP-ribosylation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase (DRAT). Several DRAT variants that are altered both in the posttranslational regulation of DRAT activity and in the ability to recognize variants of dinitrogenase reductase have been found. This correlation suggests that these two properties are biochemically connected.

scholarly journals ADP-Ribosylation Factor 1 (ARF1) Regulates Recruitment of the AP-3 Adaptor Complex to Membranes

1998 ◽  
Vol 142 (2) ◽  
pp. 391-402 ◽  
Author(s):  
Chean Eng Ooi ◽  
Esteban C. Dell'Angelica ◽  
Juan S. Bonifacino
Keyword(s):  
Membrane Trafficking ◽  
Brefeldin A ◽  
Membrane Association ◽  
Coat Proteins ◽  
Nucleotide Exchange ◽  
Membrane Recruitment ◽  
Adp Ribosylation ◽  
Adp Ribosylation Factor

Small GTP-binding proteins such as ADP- ribosylation factor 1 (ARF1) and Sar1p regulate the membrane association of coat proteins involved in intracellular membrane trafficking. ARF1 controls the clathrin coat adaptor AP-1 and the nonclathrin coat COPI, whereas Sar1p controls the nonclathrin coat COPII. In this study, we demonstrate that membrane association of the recently described AP-3 adaptor is regulated by ARF1. Association of AP-3 with membranes in vitro was enhanced by GTPγS and inhibited by brefeldin A (BFA), an inhibitor of ARF1 guanine nucleotide exchange. In addition, recombinant myristoylated ARF1 promoted association of AP-3 with membranes. The role of ARF1 in vivo was examined by assessing AP-3 subcellular localization when the intracellular level of ARF1-GTP was altered through overexpression of dominant ARF1 mutants or ARF1- GTPase-activating protein (GAP). Lowering ARF1-GTP levels resulted in redistribution of AP-3 from punctate membrane-bound structures to the cytosol as seen by immunofluorescence microscopy. In contrast, increasing ARF1-GTP levels prevented redistribution of AP-3 to the cytosol induced by BFA or energy depletion. Similar experiments with mutants of ARF5 and ARF6 showed that these other ARF family members had little or no effect on AP-3. Taken together, our results indicate that membrane recruitment of AP-3 is promoted by ARF1-GTP. This finding suggests that ARF1 is not a regulator of specific coat proteins, but rather is a ubiquitous molecular switch that acts as a transducer of diverse signals influencing coat assembly.

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