Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

The amino terminus of G protein alpha subunits is required for interaction with beta gamma

The guanine nucleotide-binding proteins (G proteins), which transduce hormonal and light signals across the plasma membrane, are heterotrimers composed of alpha, beta, and gamma subunits. Activation of G proteins by guanine nucleotides is accompanied by dissociation of the heterotrimer: G + alpha.beta.gamma in equilibrium alpha G + beta.gamma. Brain contains several G proteins of which the most abundant are alpha 39.beta.gamma and alpha 41.beta.gamma. We have used proteolysis by trypsin to study the functional domains of the alpha subunits. In the presence of guanosine 5'-(3-O-thio)triphosphate, trypsin removes a 2-kDa peptide from the amino terminus of these proteins (Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D., and Gilman, A. G. (1984) Science 226, 860-862; Winslow, J. W., Van Amsterdam, J. R., and Neer, E. J. (1986) J. Biol. Chem. 261, 7571-7579). Tryptic cleavage does not affect the GTPase activity of the truncated molecule nor the apparent Km for GTP. However, removal of the 2-kDa amino-terminal peptide prevents association of the alpha subunits with beta.gamma. Since the apparent substrate for pertussis toxin-catalyzed ADP-ribosylation is the alpha.beta.gamma heterotrimer, the trypsin-cleaved alpha subunit is not a substrate for the toxin. Digestion of the carboxyl terminus of alpha 39 with carboxypeptidase A prevents ADP-ribosylation by pertussis toxin but does not interfere with the formation of alpha 39.beta.gamma heterotrimers. We do not yet know whether the amino-terminal region of alpha 39 interacts with beta gamma directly or whether it is necessary to maintain a conformation of alpha 39 which is required for heterotrimer formation. Further studies are needed to define the nature of the contracts between alpha and beta gamma subunits since understanding the structural basis for their reversible interaction is fundamental to understanding their function.     

AMP-activated protein kinase beta subunit tethers alpha and gamma subunits via its C-terminal sequence (186-270)

AMP-activated protein kinase (AMPK) is an important metabolic stress-sensing protein kinase responsible for regulating metabolism in response to changing energy demand and nutrient supply. Mammalian AMPK is a stable alphabetagamma heterotrimer comprising a catalytic alpha and two non-catalytic subunits, beta and gamma. The beta subunit targets AMPK to membranes via an N-terminal myristoyl group and to glycogen via a mid-molecule glycogen-binding domain. Here we find that the conserved C-terminal 85-residue sequence of the beta subunit, beta1-(186-270), is sufficient to form an active AMP-dependent heterotrimer alpha1beta1-(186-270)-gamma1, whereas the 25-residue beta1 C-terminal (246-270) sequence is sufficient to bind gamma1, gamma2, or gamma3 but not the alpha subunit. Deletion of the beta C-terminal Ile-270 precludes betagamma association in the absence of the alpha subunit, but the presence of the alpha subunit or substitution of Ile-270 with Ala or Glu restores betagamma binding. Truncation of the alpha subunit reveals that beta1 binding requires the alpha1-(313-473) sequence. The conserved C-terminal 85-residue sequence of the beta subunit (90% between beta1 and beta2) is the primary alphagamma binding sequence responsible for the formation of the AMPK alphabetagamma heterotrimer.     

Immunological and biochemical differentiation of guanyl nucleotide binding proteins: interaction of Go alpha with rhodopsin, anti-Go alpha polyclonal antibodies, and a monoclonal antibody against transducin alpha subunit and Gi alpha

Guanyl nucleotide binding proteins couple agonist interaction with cell-surface receptors to an intracellular enzymatic response. In the adenylate cyclase system, inhibitory and stimulatory effects are mediated through guanyl nucleotide binding proteins, Gi and Gs, respectively. In the visual excitation complex, the photon receptor rhodopsin is linked to its target, cGMP phosphodiesterase, through transducin (Gt). Bovine brain contains another guanyl nucleotide binding protein, Go. The proteins are heterotrimers of alpha, beta, and gamma subunits; the alpha subunits catalyze receptor-stimulated GTP hydrolysis. To examine the interaction of Go alpha with beta gamma subunits and rhodopsin, the proteins were reconstituted in phosphatidylcholine vesicles. The GTPase activity of Go alpha purified from bovine brain was stimulated by photolyzed, but not dark, rhodopsin and was enhanced by bovine retinal Gt beta gamma or by rabbit liver G beta gamma. Go alpha in the presence of G beta gamma is a substrate for pertussis toxin catalyzed ADP-ribosylation; the modification was inhibited by photolyzed rhodopsin and enhanced by guanosine 5'-O-(2-thiodiphosphate). ADP-Ribosylation of Go alpha by pertussis toxin inhibited photolyzed rhodopsin-stimulated, but not basal, GTPase activity. It would appear from this and prior studies that Go alpha is similar to Gt alpha and Gi alpha; all three proteins exhibit photolyzed rhodopsin-stimulated GTPase activity, are pertussis toxin substrates, and functionally couple to Gt beta gamma. Go alpha (39K) can be distinguished from Gi alpha (41K) but not from Gt alpha (39K) by molecular weight.(ABSTRACT TRUNCATED AT 250 WORDS)     

G protein beta gamma subunits from bovine brain and retina: equivalent catalytic support of ADP-ribosylation of alpha subunits by pertussis toxin but differential interactions with Gs alpha

We have examined the ability of the beta gamma subunits of guanine nucleotide binding regulatory proteins (G proteins) to support the pertussis toxin (PT) catalyzed ADP-ribosylation of G protein alpha subunits. Substoichiometric amounts of the beta gamma complex purified from either bovine brain G proteins or the bovine retinal G protein, Gt, are sufficient to support the ADP-ribosylation of the alpha subunits of Gi (the G protein that mediates inhibition of adenylyl cyclase) and Go (a G protein of unknown function) by PT. This observation indicates that ADP-ribosylated G protein oligomers can dissociate into their respective alpha and beta gamma subunits in the absence of activating regulatory ligands, i.e., nonhydrolyzable GTP analogues or fluoride. Additionally, the catalytic support of ADP-ribosylation by bovine brain beta gamma does not require Mg2+. Although the beta gamma subunit complexes purified from bovine brain G proteins and the beta gamma complex of Gt support equally the ADP-ribosylation of alpha subunits by PT, there is a marked difference in their abilities to interact with Gs alpha. The enhancement of deactivation of fluoride-activated Gs alpha requires 25-fold more beta gamma from Gt than from brain G proteins to produce a similar response. This difference in potency of beta gamma complexes from the two sources was also observed in the ability of beta gamma to produce an increase in the activity of recombinant Gs alpha produced in Escherichia coli.     

Mutagenesis of the amino terminus of the alpha subunit of the G protein Go. In vitro characterization of alpha o beta gamma interactions.

Heterotrimeric guanine nucleotide-binding proteins are composed of alpha and beta gamma subunits and couple a variety of cell-surface receptors to intracellular enzymes or ion channels. The heterotrimer dissociates into alpha and beta gamma subunits when the alpha subunit is activated by guanine nucleoside triphosphates. Several lines of evidence show that the amino terminus of the alpha subunit is important for the interaction with the beta gamma subunit (Neer, E. J., Pulsifer, L., and Wolf, L. G. (1988) J. Biol. Chem. 263, 8996-9000; Fung, B. K.-K., and Nash, C. R. (1983) J. Biol. Chem. 258, 10503-10510). We have mutagenized the amino terminus of alpha o to dissect the relative contributions of amino-terminal myristoylation and specific amino acid sequences to subunit interaction. Wild-type and mutant alpha o cDNAs were translated in vitro in a rabbit reticulocyte lysate. All proteins were able to bind guanosine 5'-(gamma-thio)triphosphate and to achieve the necessary conformation for protection from tryptic digestion. Two assays of alpha o beta gamma interactions were used: sucrose density gradients to look for stable heterotrimer formation and ADP-ribosylation by pertussis toxin to detect weak or transient alpha o beta gamma interactions. Our results indicate that myristoylation is essential for stable heterotrimer formation, but that nonmyristoylated proteins are also capable of interacting with the beta gamma subunit. Amino acids 7-10 have an important role in alpha o beta gamma interactions whether alpha o is myristoylated or not. Deletion of this region diminishes the ability of alpha o to interact with the beta gamma subunit, but substitutions at this position indicate that other amino acids can be tolerated without affecting subunit interaction.     

Effect of monoclonal antibody binding on alpha-beta gamma subunit interactions in the rod outer segment G protein, Gt

The guanyl nucleotide binding regulatory protein of retinal rod outer segments, called Gt, that couples the photon receptor rhodopsin with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, alpha t and beta gamma t. The effect of monoclonal antibody binding to the alpha t subunit of Gt on subunit association has been investigated in the present study. It was previously shown that this monoclonal antibody, mAb 4A, blocks interactions with rhodopsin and its epitope was located within the region Arg310-Phe350 at the COOH terminus of the alpha t subunit. In this paper, we show that mAb 4A disrupts the Gt complex. Gt migrates in 5-20% linear sucrose density gradients as a monomer, with a sedimentation coefficient of 4.1 +/- 0.07 S, while in the presence of mAb 4A, the alpha t and beta gamma t subunits show sedimentation coefficients of 7.7 +/- 0.2 and 3.7 +/- 0.1 S, respectively. The beta gamma t subunit migrates with the same sedimentation rate as pure beta gamma t. Nonimmune rabbit IgG does not modify the sedimentation behavior of Gt. The Fab fragment of mAb 4A also dissociates the Gt complex, as suggested by the change of the sedimentation rate of alpha t. This effect of mAb 4A on Gt subunit association was also confirmed by immunoprecipitation studies in the presence of detergent. In the presence of detergent, subunit association is not affected, but the formation of Gt oligomers and, therefore, the nonspecific precipitation of beta gamma t subunit are reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships within the family of GTP-binding proteins isolated from bovine central nervous system

Four members of a family of GTP-binding proteins (G-proteins) which translate stimulation of extracellular receptors into regulation of intracellular enzymes were isolated from the bovine central nervous system. These proteins were examined for functional similarities and cross-reactivity with antibodies to the G-protein (transducin, Gt) from the photoreceptor system. Two proteins, Gs and Gi, can be distinguished by their respective abilities to stimulate or inhibit adenylate cyclase. The activated alpha subunits of Gt and a fourth member of the family, Go, did not affect this enzyme. Gt was shown to be unique in its ability to stimulate cGMP-dependent phosphodiesterase. While functionally diverse, the G-proteins were shown to have some common antigenic properties. Antibodies directed against the beta subunit of Gt recognize the beta 36 subunits of all preparations but not a putative second beta 35 subunit. Antibodies specific for the alpha subunit of Gt did not recognize other alpha subunits when immune blots from sodium dodecyl sulfate gels were examined. However, Go alpha, but not Gs alpha or Gi alpha, reacted strongly with the antibodies when the native subunit was spotted directly. This suggests that Go alpha and Gt alpha have homologous structural determinants. An antiserum that recognized Gt gamma did not recognize gamma subunits from other sources. These data support the proposed diversity of function and similarity of structure among the four G-proteins. The alpha and potentially gamma subunits appear to be responsible for the specificity of function.     

Expression and chain assembly of human laminin-332 in an insect cell-free translation system

Laminins are a family of large heterotrimeric glycoproteins comprising alpha, beta, and gamma chains. To determine the molecular mechanisms underlying chain assembly in vitro, we expressed human laminin-332 subunits in an insect cell-free translation system. We successfully produced the beta3-gamma2 heterodimer and the alpha3-beta3-gamma2 heterotrimer of the laminin coiled-coil (LCC) domain following co-translation of each chain. The alpha3-beta3 and the alpha3-gamma2 heterodimer were not detected, suggesting that the alpha3 chain can assemble with only beta3-gamma2 heterodimer to form a heterotrimer via disulfide bonds. These results are consistent with those of a previous report indicating that laminin chain assembly proceeds through the beta-gamma heterodimer to the alpha-beta-gamma heterotrimer in vivo. We suggest that the cell-free translation system is a valid system with which to study the mechanisms underlying laminin chain assembly.     

G(alpha)(i) controls the gating of the G protein-activated K(+) channel, GIRK.

GIRK (Kir3) channels are activated by neurotransmitters coupled to G proteins, via a direct binding of G(beta)(gamma). The role of G(alpha) subunits in GIRK gating is elusive. Here we demonstrate that G(alpha)(i) is not only a donor of G(beta)(gamma) but also regulates GIRK gating. When overexpressed in Xenopus oocytes, GIRK channels show excessive basal activity and poor activation by agonist or G(beta)(gamma). Coexpression of G(alpha)(i3) or G(alpha)(i1) restores the correct gating parameters. G(alpha)(i) acts neither as a pure G(beta)(gamma) scavenger nor as an allosteric cofactor for G(beta)(gamma). It inhibits only the basal activity without interfering with G(beta)(gamma)-induced response. Thus, GIRK is regulated, in distinct ways, by both arms of the G protein. G(alpha)(i) probably acts in its GDP bound form, alone or as a part of G(alpha)(beta)(gamma) heterotrimer.     

Site-specific antibodies directed against G protein beta and gamma subunits: effects on alpha and beta gamma subunit interaction.

Little is known about the specific domains of G protein beta and gamma subunits which interact with each other and with the alpha subunit. We used site-specific anti-peptide antibodies directed against beta and gamma subunits to investigate domains on beta and gamma subunits involved in alpha subunit interaction. Antibodies included four against the transducin (Gt) beta subunit (residues 1-10 = MS, 127-136 = KT, 256-265 = RA, and 330-340 = SW) and two against the gamma subunit (residues 2-12 = PV and 58-68 = PE). All antisera, when affinity-purified on peptide columns, yielded antibodies capable of recognizing the denatured cognate subunit on immunoblots, but only RA, SW, PV, and PE recognized native beta gamma t subunits. Affinity purification of MS and KT antisera on columns of immobilized native Gt yielded antibodies capable of recognizing native beta gamma t subunits. The functional effects of each antibody preparation on alpha t-beta gamma t interaction were assessed by assaying the ability of the preparations to immunoprecipitate beta gamma t subunits in the presence of excess alpha subunits and by testing the inhibition of beta gamma t-dependent ADP-ribosylation of alpha t-subunits catalyzed by pertussis toxin. On the basis of the results, we conclude that the domains on beta gamma t which may be directly involved in alpha t-beta gamma t interaction include the extreme amino terminus, residues 127-136 and 256-265 of beta t, and the carboxyl terminus of gamma t.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noncatalytic cGMP-binding sites of amphibian rod cGMP phosphodiesterase control interaction with its inhibitory gamma-subunits. A putative regulatory mechanism of the rod photoresponse.

The cGMP phosphodiesterase (PDE) of retinal rods plays a central role in phototransduction. Illumination leads to its activation by a rod G-protein (Gt, transducin), thus causing a decrease in intracellular cGMP concentration, closure of plasma membrane cationic channels gated by cGMP, and development of the photoresponse. The PDE holoenzyme is an alpha beta gamma 2 tetramer. The alpha- and beta-subunits each contain one catalytic and one, or possibly two, noncatalytic cGMP-binding sites. Two identical gamma-subunits serve as protein inhibitors of the enzyme. Their inhibition is removed when they bind to Gt-GTP during PDE activation. Here we report that the noncatalytic cGMP-binding sites regulate the binding of PDE alpha beta with PDE gamma and as a result determine the mechanism of PDE activation by Gt. If the noncatalytic sites are empty, Gt-GTP physically removes PDE gamma from PDE alpha beta upon activation. Alternatively, if the noncatalytic sites are occupied by cGMP, Gt-GTP releases PDE gamma inhibitory action but remains bound in a complex with the PDE heterotetramer. The kinetic parameters of activated PDE in these two cases are indistinguishable. This mechanism appears to have two implications for the physiology of photoreceptor cells. First, the tight binding of PDE gamma with PDE alpha beta when the noncatalytic sites are occupied by cGMP may be responsible for the low level of basal PDE activity observed in dark-adapted cells. Second, occupancy of the noncatalytic sites ultimately controls the rate of PDE inactivation (cf. Arshavsky, V. Yu., and Bownds, M. D. (1992) Nature 357, 416-417), for the GTPase activity that terminates PDE activity is slower when these sites are occupied and Gt stays in a complex with PDE holoenzyme. In contrast GTPase acceleration is maximal when the noncatalytic sites are empty and Gt-PDE gamma dissociates from PDE alpha beta. Because cGMP levels are known to decrease upon illumination over a concentration range corresponding to the binding constants of the noncatalytic sites, the binding might be involved in determining the lifetime of activated PDE, after a single flash and/or during dark adaptation.     

A conserved sequence immediately N-terminal to the Bateman domains in AMP-activated protein kinase gamma subunits is required for the interaction with the beta subunits

Mammalian AMP-activated protein kinase is a serine/threonine protein kinase that acts as a sensor of cellular energy status. AMP-activated protein kinase is a heterotrimer of three different subunits, i.e. alpha, beta, and gamma, with alpha being the catalytic subunit and beta and gamma having regulatory roles. Although several studies have defined different domains in alpha and beta involved in the interaction with the other subunits of the complex, little is known about the regions of the gamma subunits involved in these interactions. To study this, we have made sequential deletions from the N termini of the gamma subunit isoforms and studied the interactions with alpha and beta subunits, both by two-hybrid analysis and by co-immunoprecipitation. Our results suggest that a conserved region of 20-25 amino acids in gamma1, gamma2, and gamma3, immediately N-terminal to the Bateman domains, is required for the formation of a functional, active alphabetagamma complex. This region is required for the interaction with the beta subunits. The interaction between the alpha and gamma subunits does not require this region and occurs instead within the Bateman domains of the gamma subunit, although the alpha-gamma interaction does appear to stabilize the beta-gamma interaction. In addition, sequential deletions from the C termini of the gamma subunits indicate that deletion of any of the CBS (cystathionine beta-synthase) motifs prevents the formation of a functional complex with the alpha and beta subunits.     

Over-expression of a truncated Arabidopsis thaliana heterotrimeric G protein gamma subunit results in a phenotype similar to alpha and beta subunit knockouts

Heterotrimeric G proteins (G-proteins) are a diverse class of signal transducing proteins which have been implicated in a variety of important roles in plants. When G-proteins are activated, they dissociate into two functional subunits (alpha and the betagamma dimer) that effectively relay the signal to a multitude of effectors. In animal systems, the betagamma dimer is anchored to the plasma membrane by a prenyl group present in the gamma subunit and membrane localization has proven vital for heterotrimer function. A semi-dominant negative strategy was designed aiming to disrupt heterotrimer function in Arabidopsis thaliana (ecotype Columbia) plants by over-expressing a truncated gamma subunit lacking the isoprenylation motif (gamma()). Northern analysis shows that the levels of expression of the mutant gamma subunit in several transgenic lines (35S-gamma()) are orders of magnitude higher than that of the native subunits. In-depth characterization of the 35S-gamma() lines has been carried out, specifically focusing on a number of developmental characteristics and responses to several stimuli previously shown to be affected in alpha- and beta-deficient mutants. In all cases, the transgenic lines expressing the mutant gamma subunit behave in the same way as the alpha- and/or the beta-deficient mutants, albeit with reduced severity of the phenotype. Our data indicates that signaling from both functional subunits, alpha and the beta/gamma dimer, is disrupted in the transgenic plants. Even though physical association of the subunits has been previously reported, our research provides evidence of the functional association of alpha and beta with the gamma subunits in Arabidopsis, while also suggesting that plasma membrane localization may be critical for function of plant heterotrimeric G proteins.     

Heterotrimer formation,together with isoprenylation,is required for plasma membrane targeting of Gbetagamma

Nascent beta and gamma subunits of heterotrimeric G proteins need to be targeted to the cytoplasmic face of the plasma membrane (PM) in order to transmit signals. We show that beta(1)gamma(2) is poorly targeted to the PM and predominantly localized to endoplasmic reticulum (ER) membranes when expressed in HEK293 cells, but co-expression of a G protein alpha subunit allows strong PM localization of the beta(1)gamma(2). Furthermore, C-terminal isoprenylation of the gamma subunit is necessary but not sufficient for PM localization of beta(1)gamma(2). Isoprenylation of gamma(2) and localization of beta(1)gamma(2) to the ER occurs independently of alpha expression. Efficient PM localization of beta(1)gamma(2) in the absence of co-expressed alpha is observed when a site for palmitoylation, a putative second membrane targeting signal, is introduced into gamma(2). When a mutant of alpha(s) is targeted to mitochondria, beta(1)gamma(2) follows, consistent with an important role for alpha in promoting subcellular localization of betagamma. Furthermore, we directly demonstrate the requirement for alpha by showing that disruption of heterotrimer formation by the introduction of alpha binding mutations into beta(1) impedes PM targeting of beta(1)gamma(2). The results indicate that two membrane targeting signals, lipid modification and alpha binding, make concerted contributions to PM localization of betagamma.     

Functional molecular mapping of archaeal translation initiation factor 2

Eukaryotic and archaeal initiation factors 2 (e/aIF2) are heterotrimeric proteins (alphabetagamma) carrying methionylated initiator tRNA to the small subunit of the ribosome. The three-dimensional structure of aIF2gamma from the Archaea Pyrococcus abyssi was previously solved. This subunit forms the core of the heterotrimer. The alpha and beta subunits bind the gamma, but do not interact together. aIF2gamma shows a high resemblance with elongation factor EF1-A. In this study, we characterize the role of each subunit in the binding of the methionylated initiator tRNA. Studying various aminoacyl-tRNA ligands shows that the methionyl group is a major determinant for recognition by aIF2. aIF2gamma alone is able to specifically bind Met-tRNAiMet, although with a reduced affinity as compared with the intact trimer. Site-directed mutagenesis confirms a binding mode of the tRNA molecule similar to that observed with the elongation factor. Under our assay conditions, aIF2beta is not involved in the docking of the tRNA molecule. In contrast, aIF2alpha provides the heterotrimer its full tRNA binding affinity. Furthermore, the isolated C-domain of aIF2alpha is responsible for binding of the alpha subunit to gamma. This binding involves an idiosyncratic loop of domain 2 of aIF2gamma. Association of the C-domain of aIF2alpha to aIF2gamma is enough to retrieve the binding affinity of tRNA for aIF2. The N-terminal and central domains of aIF2alpha do not interfere with tRNA binding. However, the N-domain of aIF2alpha interacts with RNA unspecifically. Based on this property, a possible contribution of aIF2alpha to formation of a productive complex between aIF2 and the small ribosomal subunit is envisaged.     

Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment

An important challenge is to understand the functional specialization of dynein heavy chains. The ciliary outer arm dynein from Tetrahymena thermophila is a heterotrimer of three heavy chains, called alpha, beta and gamma. In order to dissect the contributions of the individual heavy chains, we used controlled urea treatment to dissociate Tetrahymena outer arm dynein into a 19S beta/gamma dimer and a 14S alpha heavy chain. The three heavy chains remained full-length and retained MgATPase activity. The beta/gamma dimer bound microtubules in an ATP-sensitive fashion. The isolated alpha heavy chain also bound microtubules, but this binding was not reversed by ATP. The 19S beta/gamma dimer and the 14S alpha heavy chain could be reconstituted into 22S dynein. The intact 22S dynein, the 19S beta/gamma dimer, and the reconstituted dynein all produced microtubule gliding motility. In contrast, the separated alpha heavy chain did not produce movement under a variety of conditions. The intact 22S dynein produced movement that was discontinuous and slower than the movement produced by the 19S dimer. We conclude that the three heavy chains of Tetrahymena outer arm dynein are functionally specialized. The alpha heavy chain may be responsible for the structural binding of dynein to the outer doublet A-tubule and/or the positioning of the beta/gamma motor domains near the surface of the microtubule track.     

Transducin-alpha C-terminal mutations prevent activation by rhodopsin: a new assay using recombinant proteins expressed in cultured cells.

We have measured the activation by recombinant rhodopsin of the alpha-subunit (alpha 1) of retinal transducin (Gt, also recombinant) using a new assay. Cultured cells are transiently transfected with DNAs encoding opsin and the three subunits of Gt (alpha t, beta 1 and gamma 1). In the microsomes of these cells, incubated with 11-cis-retinal, light causes the rapid activation of Gt, as measured by the ability of GTP gamma S to protect alpha t fragments from proteolytic degradation. The activation of Gt is also observed when all-trans-retinal is added to microsomes under constant illumination. Activation depends on both opsin and retinal. Opsin mutants with known defects in activating Gt show similar defects in this assay. alpha t mutations that mimic the corresponding mutations in the alpha-subunit of Gs also produce qualitatively similar effects in this assay. As a first step in a strategy aimed at exploring the relationships between structure and function in the interactions of receptors with G proteins, we tested mutant alpha t proteins with alanine substituted for each of the 10 amino acids at the C-terminus, a region known to be crucial for interactions with rhodopsin. Alanine substitution at four positions moderately (K341) or severely (L344, G348, L349) impairs the susceptibility of alpha 1 to activation by rhodopsin. All four mutants retain their ability to be activated by AIF-4. Two other substitutions (N343 and F350) resulted in very mild defects, while substitutions at the remaining four positions (E342, K345, D346 and C347) had no effect. In combination with previous observations, these results constrain models of the interaction of the C-terminus of alpha t with rhodopsin.     

Purification and immunochemical characterization of the major pertussis-toxin-sensitive guanine-nucleotide-binding protein of bovine-neutrophil membranes

Bovine peripheral neutrophils contain high levels of a 40-kDa pertussis toxin substrate, which was found highly enriched in a light membrane fraction upon subcellular fractionation of neutrophil homogenates. The 40-kDa pertussis toxin substrate, referred to as alpha n, was purified to near homogeneity from this fraction by sequential ion-exchange, gel-filtration and hydrophobic chromatography. Purified alpha n was shown to interact with beta gamma subunits, undergo ADP-ribosylation by pertussis toxin, and bind guanine nucleotides with high affinity. The mobility of purified alpha n on SDS/polyacrylamide gels was intermediate between those of the alpha subunits of Gi and Go, purified from bovine brain, and slightly lower than the mobility of the alpha subunit of transducin (Gt). Several polyclonal antisera against the alpha subunits of bovine Gt and Go did not react with alpha n on immunoblots. CW 6, a polyclonal antiserum reactive against the bovine alpha i, reacted only minimally with alpha n. These results suggest that the major pertussis toxin substrate of bovine neutrophils, designated Gn, is structurally different from previously identified pertussis toxin substrates and may represent a novel guanine-nucleotide-binding protein.     

Transducin activation in electropermeabilized frog rod outer segments is highly amplified, and a portion equivalent to phosphodiesterase remains membrane-bound

An electropermeabilized preparation of frog retinal rod outer segments (ROS) has been developed to examine the light sensitivity and amplification of visual transduction reactions in a minimally disturbed environment. Electropermeabilized ROS are indistinguishable from whole and osmotically intact ROS in the light microscope and retain 3-fold more protein than mechanically disrupted ROS. They differ from mechanically fragmented ROS in several respects. Illumination results in more amplified activation of the GTP-binding protein transducin (Gt) than previously observed: bleaching as little as approximately 1 rhodopsin molecule (Rho*) in every 10 disks within a single ROS activates 37,000 molecules of Gt per Rho*, equivalent to 70% of the light-activatable Gt present on a single disk face. This amplification is maintained over approximately 1 decade of light intensity but drops sharply as disk faces begin to absorb a second photon. Lower amplification is observed in fragmented ROS and derives from the fact that physical disruption of ROS causes Gt to bind GTP and elute from the membrane, thus decreasing the amount remaining and available for light activation. Illumination of electropermeabilized ROS in the presence of GTP or of the nonhydrolyzable substrate guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) causes redistribution of Gt: an amount (approximately 20 mmol/mol Rho) equivalent to the amount of inhibitory gamma subunit of phosphodiesterase (PDE) remains internal and bound to nucleotide, and the remaining activated Gt diffuses out in a manner graded with light intensity. This suggests that PDE activation by Gt alpha may not require dissociation of Gt alpha bound to the gamma subunit of PDE in a form than can elute from ROS. Two further differences between electropermeabilized and mechanically disrupted ROS are noted: the addition of ATP to electropermeabilized ROS does not affect the light sensitivity or kinetics of the GTP binding reaction, and a specificity for light-induced GTP versus GDP binding is observed.