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PubMed1985

Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site.

Pertussis toxin catalyzes the transfer of ADP-ribose from NAD to the guanine nucleotide-binding regulatory proteins Gi, Go, and transducin. Based on a partial amino acid sequence for a tryptic peptide of ADP-ribosylated transducin, asparagine had been characterized as the site of pertussis toxin-catalyzed ADP-ribosylation. Subsequently, cDNA data for the alpha subunit of transducin indicated that the putative asparagine residue was, in fact, not present in the protein. To determine the amino acid that served as the ADP-ribose acceptor, radiolabel from [adenine-U-14C]NAD was incorporated, in the presence of pertussis toxin, into the alpha subunit of transducin (0.3 mol/mol). An ADP-ribosylated, tryptic peptide was purified and fully sequenced by automated Edman degradation. The amino acid sequence, Glu-Asn 343-Leu-Lys-Asp 346-X-Gly 348-Leu-Phe, corresponds to the cDNA sequence coding the carboxyl-terminal nonapeptide, Glu 342-Phe 350, which includes by cDNA sequence cysteine at position 347. Neither Asn 343 nor Asp 346 appeared to be modified; residue 347 adhered to the sequencing resin. Cysteine, the missing residue, was eluted from the sequencing resin with acetic acid along with 76% of the peptide-associated radioactivity, half of which, presumably ADP-ribosylcysteine, eluted from an anion exchange column between NAD and ADP-ribose; the other half had a retention time corresponding to 5'-AMP. We conclude that Cys 347 and not Asn 343 or Asp 346 is the site of pertusis toxin-catalyzed ADP-ribosylation in transducin.

Adenosine Diphosphate Ribose↗
PubMed1985

[Isolation and characteristics of cyanogen bromide peptides of transducin alpha and beta subunits].

The alpha- and beta-subunits of the GTP-binding protein (transducin) from cattle retina were cleaved with cyanogen bromide. 21 peptides covering 90-100% of the amino acid sequence of the alpha- and beta-subunits were isolated from the hydrolyzate. Cyanogen bromide peptides complete or partial amino acid sequence was determined, the results were compared with those by Numa and coworkers [1] and Lochrie et al. [2] at the primary structure of the transducin alpha-subunit deduced from the nucleotide sequence of the cDNA. The structure by Lochrie is shown to differ much from the true structure of the alpha-subunit; probably, the investigators isolated cDNA, corresponding to the gene for some GTP-binding protein homologous to transducin, but not to the gene for the transducin alpha-subunit. The Numa's structure also contains an error. The final primary structure of the transducin alpha-subunit is given. The protein polypeptide chain consists of 349 amino acid residues and has an acetylmethionine residue as the N-terminal residue.

Amino Acid Sequence↗
PubMed1985

Characterization by two-dimensional peptide mapping of the gamma subunits of Ns and Ni, the regulatory proteins of adenylyl cyclase, and of transducin, the guanine nucleotide-binding protein of rod outer segments of the eye.

Ns and Ni, the regulatory proteins affecting adenylyl cyclase, and transducin, the guanine nucleotide-binding protein from rod outer segments of the eye, are structurally and functionally related proteins. Of these, the alpha subunits are between 39 and 42 kDa in mass, beta subunits are all of 35 kDa in mass, and gamma subunits are much smaller, of approximately 5-8 kDa in mass. We compared, by two-dimensional peptide mapping of iodinated peptides, the beta and gamma subunits of human erythrocyte Ns, human erythrocyte Ni, the beta gamma complex derived from purification of bovine brain N proteins, and frog and bovine eye transducins. We found that gamma subunits in human erythrocyte Ns and Ni and in bovine brain beta gamma complex are indistinguishable by this approach. In contrast, gamma subunits associated with frog and bovine transducin differed markedly between each other and from N protein-associated gamma. beta subunits, on the other hand, yielded essentially indistinguishable peptide maps regardless of whether derived from N proteins or from transducin and regardless also of species of origin: human versus bovine versus frog. These results suggest that the gamma subunit may impart functional heterogeneity of this family of proteins which is evident in the N proteins on the one hand and the transducin proteins on the other.

Animals↗
PubMed1992

Comparative analysis of the beta transducin family with identification of several new members including PWP1, a nonessential gene of Saccharomyces cerevisiae that is divergently transcribed from NMT1.

While investigating the expression of the Saccharomyces cerevisiae myristoyl-CoA:protein N-myristoyltransferase gene (NMT: E.C. 2.3.1.97) by Northern blot analysis, we observed another RNA transcript whose expression resembled that of NMT1 during meiosis and was derived from a gene located less than 1 kb immediately upstream of NMT1. This new gene, designated PWP1 (for periodic tryptophan protein), is divergently transcribed from NMT1 and encodes a 576-residue protein. Null mutants of PWP1 are viable, but their growth is severely retarded and steady-state levels of several cellular proteins (including at least two proteins that label with exogenous [3H]myristic acid) are drastically reduced. New methods for database searching and assessing the statistical significance of sequence similarities identify PWP1 as a member of the beta-transducin protein superfamily. Two other previously unrecognized beta-transducin-like proteins (S. cerevisiae MAK11 and D. discoideum AAC3) were also identified, and an unexpectedly high degree of sequence homology was found between a Chlamydomonas beta-like polypeptide and the C12.3 gene of chickens. A systematic and quantitative comparative analysis resulted in classifying all beta-transducin-like sequences into 11 nonorthologous families. Based on specific sequence attributes, however, not all beta-transducin-like sequences are expected to be functionally similar, and quantitative criteria for inferring functional analogies are discussed. Possible roles of repetitive tryptophan residues in proteins are also considered.

Acyltransferases↗
PubMed1999

Interaction of the retinal G-protein transducin with uracil nucleotides.

Little is known about the interaction of pyrimidine nucleotides with G-proteins. Here we report that under experimental conditions that exclude transphosphorylation reactions, nucleoside 5'-triphosphates inhibited transducin-catalyzed GTP hydrolysis in the order of potency guanosine 5'-[gamma-thio]triphosphate > GTP > guanosine 5'-[beta,gamma-imido]triphosphate > uridine 5'-[gamma-thio]triphosphate > UTP > CTP. Nucleoside 5'-diphosphates inhibited GTP hydrolysis in the order of potency GDP approximately guanosine 5'-[beta-thio]thiodiphosphate > uridine 5'-[beta-thio]diphosphate >> UDP (no effect). UTP inhibited GTP hydrolysis competitively, indicative for nucleotide binding to the same site. Uracil nucleotides had a distinct activity profile with respect to disruption of the transitory complex between photoexcited rhodopsin and nucleotide-free transducin. We conclude that (i) uracil nucleotides bind to transducin-alpha with lower affinity than the corresponding guanine nucleotides, (ii) phosphorothioate modification of uracil nucleotides increases their affinity for transducin, and (iii) uracil nucleotides induce conformational changes in G-proteins that are different from the conformational changes induced by guanine nucleotides.

Animals↗
PubMed1989

Evidence for oligomeric forms of transducins alpha subunit: formation of intermolecular alpha-alpha disulfide linkages.

Transducin, the retinal G-protein, is a heterotrimeric protein composed of alpha, beta and gamma subunits. Intermolecular disulfide linkages between the alpha-subunits of transducin molecules are spontaneously formed when the purified G-protein is placed in a non-reducing buffer system. The beta and gamma subunits do not participate in the intermolecular disulfide bridge formation. The alpha-alpha subunit disulfide bonds result in the inhibition of transducin activation by bleached rhodopsin which is restored by reducing the disulfides with dithiothreitol. The trapping of oligomers by disulfide bond formation provides physical evidence for specific intermolecular interactions between alpha-subunits of transducin.

Animals↗
PubMed1986

Localization and quantitation of opsin and transducin mRNAs in bovine retina by in situ hybridization histochemistry.

Oligodeoxynucleotide probes complementary to a portion of bovine opsin mRNA and transducin mRNA were used for in situ hybridization histochemistry. Within the retina, only photoreceptors expressed mRNAs detectable with these probes, and the majority of both mRNAs were in photoreceptor inner segments. More opsin mRNA was detected than transducin mRNA. In the inner segments 0.54 +/- 0.05 copies/micron3 of opsin mRNA and 0.34 +/- 0.05 copies/micron3 of transducin mRNA were detected. In the outer nuclear layer, 0.39 +/- 0.06 copies/micron3 of opsin mRNA and 0.27 +/- 0.04 copies/micron3 of transducin mRNA were detected.

Animals↗
PubMed1987

On the role of transducin GTPase in the quenching of a phosphodiesterase cascade of vision.

The rate of GTP hydrolysis in the active site of transducin and that of the release of the phosphate thus formed have been measured. The former step has been found to be a rate-limiting one. The rate constant for GTP hydrolysis is equal to 0.027 s-1 at 23 degrees C, and 0.07 s-1 at 37 degrees C. Besides, it has been shown that the rate of GTPase reaction on the transducin alpha-subunit does not depend on the concentration of a complex of transducin beta- and gamma-subunits or on the presence of cGMP phosphodiesterase and a 48 kDa protein from rod outer segments. According to the results, GTP hydrolysis on transducin proceeds too slowly to account for the rapid quenching of a phosphodiesterase cascade in rod outer segments.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1990

Binding of GTP to transducin is not inhibited by arrestin and phosphorylated rhodopsin.

In the presence of a photobleaching intermediate of unphosphorylated or phosphorylated rhodopsin (Rh*), the binding of GppNHp to transducin was measured with or without arrestin for elucidation of the shut-off mechanism of the visual transduction process in bovine rod outer segments. The ability of Rh* to catalyze the formation of the transducin-GppNHp complex in the absence of arrestin was independent of the degree of phosphorylation of Rh*. Furthermore, the catalyzing ability of the phosphorylated Rh* was not reduced by the addition of arrestin. These observations indicate that the interaction between phosphorylated Rh* and transducin was not inhibited by arrestin. Thus, the hypothesis was not supported that the PDE shut-off process is a simple competition between transducin and arrestin for binding to phosphorylated Rh*.

Animals↗
PubMed1991

Choleratoxin ADP-ribosylates transducin only when it is bound to photoexcited rhodopsin and depleted of its nucleotide.

The sensitivity of transducin (T) to choleratoxin (CT) in retinal cells depends on illumination and on the presence of GTP or analogs. Low concentrations of GPP-NH-P or GPP-CH2-P increase ADP-ribosylation while GTP gamma S inhibits it. We show that GTP analogs permanently activate an ADP-ribosylating factor (ARF) which mediates CT action on retinal cell membranes: when transducin-depleted membranes were pre-activated by GTP analogs, re-added transducin became sensitive to CT in the absence of nucleotide, and presence of photoexcited rhodopsin (R*). Any subsequent G-nucleotide addition (even GDP) decreased ADP-ribosylation. Thus nucleotide-free transducin molecule in R*-Tempty complex is the CT substrate.

Adenosine Diphosphate Ribose↗
PubMed1986

Models for quick activation of transducin and spontaneous deactivation of activated rhodopsin in rod outer segments.

Serial-parallel activation (S-P) and parallel-parallel activation (P-P) models had been proposed to account for the quick activation of transmitter molecules (transducin) in the visual transduction process in rod outer segments. In these models, unbleached rhodopsin molecules are activated by bleached rhodopsin molecules, and both bleached and activated rhodopsin molecules activate transducin. This activation mechanism enables bleached rhodopsin molecules to transfer the information of photon detection quickly to transducin. In the previous models, however, the rhodopsin photoisomerization process and the deactivation process of activated rhodopsin molecules were excluded. In the present study, these two processes are incorporated into the models. The deactivation process of activated rhodopsin molecules is assumed to proceed spontaneously. It is presented that, in spite of the incorporation of these two processes, the present S-P and P-P models can account for the quick activation of transducin in rod outer segments.

Animals↗
PubMed1998

Identification of a new intermediate state that binds but not activates transducin in the bleaching process of bovine rhodopsin.

Using time-resolved low-temperature spectroscopy, we have examined whether or not bovine rhodopsin has a unique transducin-binding state, meta Ib, previously detected from chicken rhodopsin. Unlike chicken meta Ib, bovine meta Ib was detected only by detailed kinetics analysis of the bleaching process, but it was stabilized by transducin and visualized in the observed spectral changes. From the effect of GTPgammaS, it was revealed that meta Ib induced no GDP-GTP exchange reaction in transducin. Thus meta Ib is a common intermediate of vertebrate rhodopsin and transducin is activated in two steps by meta Ib and meta II.

Animals↗
PubMed2002

Signal-dependent translocation of transducin, RGS9-1-Gbeta5L complex, and arrestin to detergent-resistant membrane rafts in photoreceptors.

Many lines of evidence show that membranes contain microdomains, "lipid rafts", that are different from the rest of the membrane in specific lipid and protein composition. In several biological systems, they were shown to be necessary for trafficking and signal transduction. Here, we investigate if lipid rafts have a role in the regulation of the G protein-mediated pathway underlying vertebrate phototransduction. Photoreceptor membranes contain detergent-resistant membrane (DRM) rafts. Rhodopsin and cGMP phosphodiesterase are found in raft and nonraft portions of the membrane; guanylate cyclase is found exclusively in the raft. Distribution of these proteins does not change in the light or dark. In contrast, the G protein transducin, the RGS9-1-Gbeta5L complex, and the p44 isoform of arrestin undergo dramatic translocation to the raft upon illumination. Phosphorylation of RGS9-1 occurs exclusively in the raft. GTPgammaS or pertussis toxin prevent the light-mediated translocation of transducin and RGS9-1, whereas AlF(minus sign)(4) causes both proteins to move to the raft in the dark. This shows that the Galphat-RGS9-1-Gbeta5L complex has the highest affinity to rafts in the transition state of the GTPase. GTPgammaS binds to transducin at a significantly slower rate in the raft, indicating that this translocation results in a reduced rhodopsin-transducin coupling. Thus, an external signal can rearrange components of a G protein pathway in specific domains of the cell membrane, changing its signaling properties. These findings could reveal a novel mechanism utilized by the cells for regulation of G protein-mediated signal transduction.

Animals↗
PubMed1993

GTP hydrolysis by purified alpha-subunit of transducin and its complex with the cyclic GMP phosphodiesterase inhibitor.

The single-turn GTP hydrolysis by isolated and soluble transducin has been time-resolved using a rapid flow filtration technique which takes advantage of the GTP-requiring detachment of transducin alpha-subunits (T alpha) from photoactivated rhodopsin (R*). Illuminated rod outer segment (ROS) fragments to which holo-transducin is tightly bound are retained on a syringe filter that is washed continuously with a buffer containing no GTP. When the flow is switched to a buffer with GTP, T alpha GTP is specifically eluted and injected into a cuvette where GTP hydrolysis is monitored via the associated change in the T alpha intrinsic tryptophan fluorescence. Low concentrations of GTP elute the complete pool of T alpha from the filter-retained ROS fragments in less than 1 s. This directly demonstrates that, upon GTP loading, T alpha becomes instantly soluble in physiological buffers (120 mM KC1 and 2 mM MgCl2). When all alone, T alpha hydrolyzes its bound GTP in 21 +/- 1 s (1/e time at 25 degrees C). Replacing chloride by other anions increases the GTPase rate by 2-fold. The K50 for chloride inhibition of GTPase is approximately 2 mM. Slower GTP hydrolysis is observed for cholera-toxin-modified transducin or when GTP alpha S (Sp) replaces GTP in the eluting buffer. No signal is observed when GTP gamma S is used. The GTPase rate is unaffected when T alpha GTP binds to the inhibitory subunit (PDE gamma) of the cGMP phosphodiesterase (PDE), although this binding is fast and of high affinity.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1993

Light-dependent transducin activation by an ultraviolet-absorbing rhodopsin mutant.

The photoactivation pathway of an ultraviolet-absorbing rhodopsin mutant was studied. The mutant pigment, in which the retinylidene Schiff base counterion, Glu113, was replaced by glutamine (E113Q), was known to exist in a pH-dependent equilibrium between spectral forms absorbing at about 380 and 490 nm. The 380-nm form contains an unprotonated Schiff base chromophore linkage, whereas the 490-nm form contains a protonated Schiff base chromophore linkage. The role of the Schiff base proton in photoactivation was investigated by measuring transducin activation as a function of photoactivation wavelength. The transducin activation action spectra of rhodopsin and of mutant E113Q were found to be very similar to their UV-visible absorption spectra. Thus, the 380-nm UV form of the mutant E113Q could be activated directly by UV light to catalyze nucleotide exchange by transducin. The quantum efficiency of photoactivation of the UV-absorbing form of E113Q was similar to that of its visible-absorbing form. These results show that the presence of a protonated Schiff base in the ground state is not necessarily required for efficient photoactivation of visual pigments. They support the hypothesis that the key role of the protonated Schiff base in visible-absorbing pigments is to stabilize the ground state and to allow absorbance at wavelengths above about 420 nm. The findings are also consistent with transducin activation studies of mutant apoproteins regenerated with all-trans-retinal, or of mutant apoproteins alone, suggesting that the active state of rhodopsin can be formed via a number of pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Modulation of the GTPase activity of transducin. Kinetic studies of reconstituted systems.

We seek to define the influence of retinal cGMP phosphodiesterase (PDE) on the GTPase activity of transducin (T). A novel stopped-flow/fast filtration apparatus [Antonny, B., et al. (1993) Biochemistry 32, 8646-8653] is used to deliver T alpha GTP free of rod outer segment (ROS) membranes to a suspension of phospholipid vesicles bearing holoPDE. As measured by a pH electrode, the decay of cGMP hydrolysis from these samples, which contain no other proteins but T alpha and holoPDE, requires GTP hydrolysis and occurs in 40 s. The addition of T beta gamma to the vesicles does not accelerate this deactivation. When ROS membranes are urea-stripped, reconstituted with transducin + holoPDE, and illuminated, the injection of an amount of GTP that is substoichiometric to holoPDE gives a cGMP hydrolysis pulse that lasts for 30 s. However, the same reconstitution performed with ROS stripped by extensive dilution in isotonic buffer results in a deactivation time of only 8 s, which resembles the 7 s observed with native ROSs. With these isotonically stripped ROSs, when GTP injection comes after a first injection with GTP gamma S, the cGMP hydrolysis pulse is lengthened and lasts for 17 s; with urea-washed ROS, no such lengthening is observed. These results clearly demonstrate that holoPDE by itself cannot enhance the GTPase activity of transducin, even when the two proteins are localized on a membrane surface. Instead, they point to the existence of a membrane-bound, urea-sensitive protein factor that activates the GTPase of T alpha in the transducin-holoPDE complex.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1987

A novel complex from bovine visual cells of a 33,000-dalton phosphoprotein with beta- and gamma-transducin: purification and subunit structure.

Photoreceptors of mammalian retinas contain a 33-kDa (33K) protein that is phosphorylated, in vitro, by cyclic nucleotide dependent protein kinases. The 33K protein is phosphorylated in the dark, in situ, and dephosphorylated upon illumination. The soluble 33K protein from bovine retinas has been purified to near homogeneity by extraction at pH 5.7 and chromatography on ion-exchange, gel filtration, and hydroxylapatite columns. In the native conformation, the 33K protein is associated with a 37-kDa (37K) and a 10-kDa (10K) protein, forming a trimeric complex with a sedimentation coefficient of 4.9 S and an apparent molecular mass of 77 kDa. The 33K protein can be dissociated from the 37K/10K complex by centrifugation in the presence of high pH and high salt; the subunits reassociate to form the trimeric complex upon recentrifugation in an isotonic buffer with neutral pH. The 33K protein is phosphorylated rapidly by exogenous kinase, in vitro, whereas the 37K and 10K subunits remain unphosphorylated. The 37K and 10K subunits cross-react with antibodies prepared against the beta- and gamma-subunits, respectively, of bovine transducin, indicating that the 37K and 10K subunits are immunologically identical with beta- and gamma-transducin, respectively. No immuno-cross-reactivity was observed between the 33K protein and an antibody against the alpha-subunit of bovine transducin. The 33K-beta-/gamma-transducin complex exhibits striking similarity to transducin in its subunit structure and mode of subunit interaction, suggesting it may play an important role in the metabolism and function of rod photoreceptor cells.

Animals↗
PubMed2001

A conformational switch in the inhibitory gamma-subunit of PDE6 upon enzyme activation by transducin.

In response to light, a photoreceptor G protein, transducin, activates cGMP-phosphodiesterase (PDE6) by displacing the inhibitory gamma-subunits (Pgamma) from the enzyme's catalytic sites. Evidence suggests that the activation of PDE6 involves a conformational change of the key inhibitory C-terminal domain of Pgamma. In this study, the C-terminal region of Pgamma, Pgamma-73-85, has been targeted for Ala-scanning mutagenesis to identify the point-to-point interactions between Pgamma and the PDE6 catalytic subunits and to probe the nature of the conformational change. Pgamma mutants were tested for their ability to inhibit PDE6 and a chimeric PDE5-conePDE6 enzyme containing the Pgamma C-terminus-binding site of cone PDE. This analysis has revealed that in addition to previously characterized Ile86 and Ile87, important inhibitory contact residues of Pgamma include Asn74, His75, and Leu78. The patterns of mutant PDE5-conePDE6 enzyme inhibition suggest the interaction between the PgammaAsn74/His75 sequence and Met758 of the cone PDE6alpha' catalytic subunit. This interaction, and the interaction between the PgammaIle86/Ile87 and PDE6alpha'Phe777/Phe781 residues, is most consistent with an alpha-helical structure of the Pgamma C-terminus. The analysis of activation of PDE6 enzymes containing Pgamma mutants with Ala-substituted transducin-contact residues demonstrated the critical role of PgammaLeu76. Accordingly, we hypothesize that the initial step in PDE6 activation involves an interaction of transducin-alpha with PgammaLeu76. This interaction introduces a bend into the alpha-helical structure of the Pgamma C-terminus, allowing transducin-alpha to further twist the C-terminus thereby uncovering the catalytic pocket of PDE6.

3',5'-Cyclic-GMP Phosphodiesterases↗