cGMP binding to noncatalytic sites on mammalian rod photoreceptor phosphodiesterase is regulated by binding of its gamma and delta subunits.

The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, alpha beta gamma2) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, alpha beta gamma2 delta) can release and bind cGMP at both noncatalytic sites; the delta subunit is likely responsible for this difference in cGMP exchange rates. Removal of the delta and/or gamma subunits yields a catalytic alphabeta dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (KD >1 microM); cGMP binding affinity at the second site (KD approximately 60 nM) is reduced 3-4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of gamma subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the gamma subunit with the alphabeta dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells.     

In vivo differential prenylation of retinal cyclic GMP phosphodiesterase catalytic subunits.

A number of phototransducing proteins in vertebrate photoreceptors contain a carboxyl terminal -CXXX motif (where C = cysteine and X = any amino acid), known to be a signal sequence for their post-translational prenylation and carboxyl methylation. To study the roles of these modifications in the visual excitation process, we have utilized an intravitreal injection method to radiolabel the prenylated proteins of rat retinas in vivo. We showed that two of the major prenylated polypeptides in the rod outer segments are the PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase PDE alpha and PDE beta subunits of cyclic GMP phosphodiesterase (PDE). By chromatographic analyses of the amino acid constituents generated by exhaustive proteolysis of PDE alpha and PDE beta, we further demonstrated that they are differentially prenylated by farnesylation and geranylgeranylation, respectively. While a number of proteins ending with the -CXXX sequence have already been reported to possess either a farnesyl or a geranylgeranyl group, PDE is the first enzyme shown to be modified by both types of prenyl groups. The prenyl modification of PDE most likely plays a major role in membrane attachment and in correctly positioning the PDE molecule for phototransduction.     

The cGMP phosphodiesterase-transducin complex of retinal rods. Membrane binding and subunits interactions.

cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is composed of two catalytic subunits (PDE alpha and PDE beta) and two identical inhibitory subunits (PDE gamma). Native PDE alpha beta gamma 2 is peripherally bound to the membranes of ROS discs. We studied quantitatively its partition between soluble and membrane-bound fractions in ROS homogenates. In the presence of its activator, the alpha-subunit of transducin loaded with a triphosphate guanine nucleotide (T alpha*), PDE displayed a greatly enhanced membrane binding. Neither the purified PDE gamma.T alpha* complex, nor the PDE alpha beta and PDE alpha beta gamma forms of active PDE, showed a membrane binding comparable to that of PDE alpha beta gamma 2 in the presence of T alpha*. The T alpha*-activated PDE is therefore an undissociated complex tightly bound to the ROS membranes. Using limited proteolysis, we showed that the membrane anchoring of the whole complex implies not only PDE (mainly by the C terminus of PDE beta) but also both termini of T alpha*. The membrane binding of the purified PDE alpha beta species was also enhanced in the presence of T alpha*; a direct link would therefore exist between the activator and the catalytic subunits. From this work emerges a plausible structural model of the T alpha*-activated PDE, with its internal interactions and its sites of anchoring into the ROS membrane.     

Identification of the retinal cyclic GMP phosphodiesterase inhibitory gamma-subunit interaction sites on the catalytic alpha-subunit.

Retinal rod outer segment phosphodiesterase (PDE) consists of two similar catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). A trypsin-activated soluble PDE exhibiting the ability to be reinhibited by PDE gamma was shown by peptide antisera to retain both N and C termini. Synthetic peptides corresponding to residues 16-30, 78-90, 389-403, and 535-563 of PDE alpha used in a PDE activity assay with trypsin-activated PDE partially prevented inhibition by exogenous PDE gamma; however, only competitions by peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90) were concentration-dependent below 100 nmol of peptide. Binding studies using radio-immunoassays and PDE alpha peptides confirmed that peptides 16-30 and 78-90 (corresponding to PDE alpha 16-30 and 78-90, respectively) were able to bind PDE gamma. Additionally, peptides corresponding to the PDE alpha region 453-534 bound PDE gamma in the binding assay. This suggests that several regions on PDE alpha interact with the PDE gamma inhibitor. While some regions may be involved in binding to PDE gamma, other sites may be involved in PDE gamma inhibition of catalytic activity. Our results suggest that the major regions of PDE alpha that interact with PDE gamma reside within the N terminus (16-30 and 78-90), with weaker interaction regions within or near the hypothesized catalytic domain (453-563). Sequence analysis of three retinal phosphodiesterases (rod outer segment alpha, beta, and cone outer segment alpha') revealed the highest region of dissimilarity in the N and C termini.     

Activation and solubilization of the retinal cGMP-specific phosphodiesterase by limited proteolysis. Role of the C-terminal domain of the beta-subunit.

The cGMP-specific phosphodiesterase (PDE) of vertebrate retinal rod outer segments (ROS) is a peripheral enzyme activated in vivo by transducin. In vitro artificial activation can be achieved using trypsin. This was described as resulting from degradation of the inhibitory gamma subunit (2 copies/PDE molecule), leaving intact the alpha beta catalytic core. It was, however, observed that trypsin could induce the release of PDE (or solubilization) from the ROS membranes before its activation [Wensel, T. G. & Stryer, L. (1986) Proteins Struct. Funct. Genet. 1, 90-99]. Studying the time course of this solubilization, we were able to purify a trypsin-solubilized PDE still completely inhibited (i.e. with its two gamma subunits bound). The tryptic solubilization of PDE is therefore complete before any functional degradation of the gamma subunits occurs. It was recently suggested that this solubilization could coincide with the cleavage of a C-terminal fragment of the alpha subunit, which can be labeled by methylation of a terminal cysteine residue [Ong, O. C., Ota, I. M., Clarke, S. & Fung, B. K. K. (1989) Proc. Natl Acad. Sci. USA 86, 9238-9242]. We present the following evidence indicating that the C-terminus of the PDE beta subunit is mainly responsible for PDE anchorage to the ROS membrane. (a) The trypsin-solubilized PDE alpha beta gamma 2 has intact blocked N-termini. (b) It is still methylated on PDE alpha. (c) The C-terminus of PDE beta can also be labeled by methylation and its tryptic cleavage coincides well with the PDE solubilization. (d) Sequential cleavage of the alpha and beta polypeptides can also be detected by high-resolution gel electrophoresis: the first cleavage appears on the beta subunit and is completed when cleavage of the alpha subunit begins. The time course for cleavage of the gamma subunits appears to be slower than for the beta subunit and comparable to that of the alpha subunit. Upon longer trypsinization, a 70-kDa polypeptide appears which seems to be a degradation product of PDE beta. Gel-filtration analysis, however, shows that this 70-kDa fragment does not dissociate from the catalytic core.     

Heterogeneity of the retinal G-protein transducin from frog rod photoreceptors. Biochemical identification and characterization of new subunits.

Transducin, a retinal G-protein, has been shown to exist as heterotrimers of alpha (39,000), beta (36,000), and gamma (approximately 7,000) subunits. Blue Sepharose CL-6B column chromatography of a transducin preparation extracted with a metal-free, low salt buffer containing GTP showed three distinct alpha and two distinct beta gamma activities in frog (Rana catesbeiana) rod outer segment. The binding of a hydrolysis-resistant GTP analog in these alpha fractions was proportional to the amount of the M(r) 39,000 protein. The first alpha was eluted in a complex with an inhibitory subunit of cGMP phosphodiesterase, but alpha subunits in the second and the third fractions were not complexed with any proteins. Two-dimensional gel electrophoresis and characterization with regard to the interaction with the inhibitory subunit of cGMP phosphodiesterase suggested that the first and the second alpha s were the same protein; however, the third alpha showed different characters as follows. We designated alpha in the first two fractions as alpha 1, and alpha in the third fraction as alpha 2. Nonlinear regression analysis for the binding of a hydrolysis-resistant GTP analog to both alpha subunits revealed a single class of GTP binding sites with an apparent stoichiometry of 1 mol of GTP/mol of alpha. Compared with alpha 1, alpha 2 required larger amounts of rhodopsin and beta gamma for the binding of a hydrolysis-resistant GTP analog. alpha 2 also showed less binding with the inhibitory subunit of cGMP phosphodiesterase. Both alpha 1 and alpha 2 complexed with beta gamma or beta delta (described below) were substrates for pertussis toxin-dependent ADP-ribosylation. The protein profiles of two beta gamma fractions revealed that the main fraction was composed of a beta gamma complex; however, the second active fraction was composed of beta complexed with delta (M(r) 12,000). Compared with beta gamma, beta delta stimulated GTP binding to alpha 1 at approximately 10-fold higher concentration. Two-dimensional gel electrophoresis revealed five beta and two gamma isoforms in beta gamma. Only one beta isoform was present in beta delta. The diversity of transducin subunits may reflect different signaling pathways in visual signal transduction.     

Characterization of transducin from bovine retinal rod outer segments. Mechanism and effects of cholera toxin-catalyzed ADP-ribosylation

Transducin, a guanine nucleotide-binding protein consisting of two subunits (T alpha and T beta gamma), mediates the signal coupling between rhodopsin and a membrane-bound cyclic GMP phosphodiesterase in retinal rod outer segments. The T alpha subunit is an activator of the phosphodiesterase, and the function of the T beta gamma subunit is to physically link T alpha with photolyzed rhodopsin. In this study, the mechanism of cholera toxin-catalyzed ADP-ribosylation of T alpha has been examined in a reconstituted system consisting of purified transducin and stripped rod outer segment membranes. Limited proteolysis of the labeled T alpha with trypsin indicated that the inserted ADP-ribose is located exclusively on a single proteolytic fragment with an apparent molecular weight of 23,000. Maximal incorporation of ADP-ribose was achieved when guanosine 5'-(beta, gamma-imido)triphosphate (Gpp(NH)p) and T beta gamma were present at concentrations equal to that of T alpha and when rhodopsin was continuously irradiated with visible light in the 400-500 nm region. The stimulating effect of illumination was related to the direct interaction of the retinal chromophore with opsin. These findings strongly suggest that a transient protein complex consisting of T alpha X Gpp(NH)p, T beta gamma, and a photointermediate of rhodopsin is the required substrate for cholera toxin. Single turnover kinetic measurements demonstrated that the ADP-ribosylation of T alpha coincided with the appearance of a population of transducin molecules having a very slow rate of GTP hydrolysis. The hydrolysis rate of the bound GTP for this population was 1.1 X 10(-3)/s, which was 22-fold slower than the rate for the unmodified transducin.     

Evaluation of the 17-kDa prenyl-binding protein as a regulatory protein for phototransduction in retinal photoreceptors

The mammalian rod photoreceptor phosphodiesterase (PDE6) holoenzyme is isolated in both a membrane-associated and a soluble form. Membrane binding is a consequence of prenylation of PDE6 catalytic subunits, whereas soluble PDE6 is purified with a 17-kDa prenyl-binding protein (PDEdelta) tightly bound. This protein, here termed PrBP/delta, has been hypothesized to reduce activation of PDE6 by transducin, thereby desensitizing the photoresponse. To test the potential role of PrBP/delta in regulating phototransduction, we examined the abundance, localization, and potential binding partners of PrBP/delta in retina and in purified rod outer segment (ROS) suspensions whose physiological and biochemical properties are well characterized. The amphibian homologue of PrBP/delta was cloned and sequenced and found to have 82% amino acid sequence identity with mammalian PrBP/delta. In contrast to bovine ROS, all of the PDE6 in purified frog ROS is membrane-associated. However, addition of recombinant frog PrBP/delta can solubilize PDE6 and prevent its activation by transducin. PrBP/delta also binds other prenylated photoreceptor proteins in vitro, including opsin kinase (GRK1/GRK7) and rab8. Quantitative immunoblot analysis of the PrBP/delta content of purified ROS reveals insufficient amounts of PrBP/delta (<0.1 PrBP/delta per PDE6) to serve as a subunit of PDE6 in either mammalian or amphibian photoreceptors. The immunolocalization of PrBP/delta in frog and bovine retina shows greatest PrBP/delta immunolabeling outside the photoreceptor cell layer. Within photoreceptors, only the inner segments of frog double cones are strongly labeled, whereas bovine photoreceptors reveal more PrBP/delta labeling near the junction of the inner and outer segments (connecting cilium) of photoreceptors. Together, these results rule out PrBP/delta as a PDE6 subunit and implicate PrBP/delta in the transport and membrane targeting of prenylated proteins (including PDE6) from their site of synthesis in the inner segment to their final destination in the outer segment of rods and cones.     

Comparison of the phosphodiesterase inhibitory subunit interactions of frog and bovine rod outer segments.

The rod outer segments of the bovine and frog retina possess a cyclic GMP phosphodiesterase (PDE) that is composed of two larger subunits, alpha and beta (P alpha beta), which contain the catalytic activity and a smaller gamma (P gamma) subunit which inhibits the catalytic activity. We studied the binding of P gamma to P alpha beta in both the bovine and frog rod outer segment membranes. Analysis of these data indicates that there are two classes of P gamma binding sites per P alpha beta in both species. The activation of PDE by the guanosine 5'-[gamma-thio]triphosphate form of the alpha subunit of transducin, T alpha.GTP gamma S, was also studied. These data indicate that the two classes of P gamma binding sites contribute to the formation of two classes of binding sites for T alpha.GTP gamma S. We demonstrate solubilization of a portion of the P gamma by T alpha.GTP gamma S in both species. There is also present, in both species, a second class of P gamma which is not solubilized even when it is dissociated from its inhibitory site on P alpha beta by T alpha.GTP gamma S. The amount of full PDE activity which results from release of the solubilizable P gamma is about 50% in the frog PDE but only approx. 17% in the bovine PDE. We also show that activation of frog rod outer segment PDE by trypsin treatment releases the PDE from the membranes. This type of release by trypsin has already been demonstrated in bovine rod outer segments [Wensel & Stryer (1986) Proteins: Struct. Funct. Genet. 1, 90-99].     

Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits

The subunits of phosphorylase kinase are separated and isolated in high yield by gel filtration chromatography in pH 3.3 phosphate buffer containing 8 M urea. Three protein peaks are obtained: the alpha and beta subunits coelute in the first, whereas the gamma and delta subunits are separate peaks. Upon dilution of the denaturant, catalytic activity reappears, associated only with the gamma subunit. As has been previously observed (Kee, S.M., and Graves, D.J. (1986) J. Biol. Chem. 261, 4732-4737), addition of calmodulin dramatically stimulates the reactivation of gamma. Inclusion of increasing amounts of the alpha/beta subunit mixture in the renaturation progressively decreases the activity of the renatured gamma or gamma-calmodulin. This inhibition by alpha/beta is likely due to specific interactions with the gamma subunit because the inhibition is less at pH 8.2 than at pH 6.8 and less when equivalent amounts of phosphorylated alpha/beta subunits are used (both alkaline pH and phosphorylation are known to stimulate the activity of the holoenzyme). These results suggest that the role of either the alpha or beta subunits, or perhaps both, in the nonactivated (alpha 2 beta 2 gamma 2 delta 2)2 complex of phosphorylase kinase is to suppress the activity of the gamma subunit and that activation of the enzyme, by phosphorylation for instance, is due to deinhibition caused by release of this quaternary constraint by alpha and/or beta upon gamma.     

A soluble form of bovine rod photoreceptor phosphodiesterase has a novel 15-kDa subunit

A substantial fraction (20-30%) of the bovine rod outer segment phosphodiesterase (PDE) activity is not associated with outer segment membranes prepared with buffers of moderate ionic strength; this PDE activity appears to represent a distinct, soluble isozyme. Although this PDE isozyme can be demonstrated to be present in sealed rod outer segments, it is discarded from most standard rod outer segment preparations. A method was developed that allowed the rapid purification of the soluble rod PDE by 2600-fold, to apparent homogeneity, using a monoclonal antibody column (ROS-1a). The soluble rod PDE isozyme has a novel Mr = 15,000 subunit (delta) in addition to subunits of Mr = 88,000 (alpha sol), 84,000 (beta sol), and 11,000 (gamma sol). The delta subunit comigrates with and may be identical to the cone PDE 15-kDa subunit. The small subunits of the soluble rod PDE and the membrane-associated rod PDE were isolated by reverse-phase chromatography. The gamma sol subunit was a potent inhibitor of trypsin-activated rod PDE, inhibiting 50% of 1 pM PDE activity at a concentration of 11 pM. This concentration was similar to that observed for the gamma subunit of the membrane-associated rod PDE. The purified delta subunit did not appear to affect PDE activity; this subunit was, however, unusually difficult to keep in solution. All of the kinetic and physical properties of the soluble rod PDE tested thus far are similar to those of the membrane-associated form, except for the presence of the delta subunit, suggesting that this unique subunit could mediate the solubility of the soluble rod PDE and the cone PDE in the intact photoreceptor.     

Functional regions of the inhibitory subunit of retinal rod cGMP phosphodiesterase identified by site-specific mutagenesis and fluorescence spectroscopy.

In the dark, the activity of the cGMP phosphodiesterase (PDE) of retinal rod outer segments is held in check by its two inhibitory gamma subunits. Following illumination, gamma is rapidly removed from its inhibitory site by transducin, the G-protein of the visual system. In order to probe the functional roles of specific regions in the PDE gamma primary sequence, 10 variants of PDE gamma have been produced by site-specific mutagenesis and expression in bacteria and their properties compared to those of protein containing the wild-type bovine PDE gamma amino acid sequence. Three questions were asked about each mutant: What is its affinity for the alpha beta catalytic subunit of PDE? Does it inhibit catalytic activity? If so, can transducin relieve this inhibition? Binding to PDE alpha beta was determined directly using fluorescein-labeled gamma by measuring the increase in emission anisotropy that occurs when gamma binds to alpha beta. Inhibition of PDE alpha beta was measured by reconstitution of the gamma variants with gamma-free PDE generated by limited digestion with trypsin or endoproteinase Arg-C. Unlike trypsin, the latter enzyme did not remove PDE's ability to bind membranes and be activated by transducin, so that transducin activation of PDE containing specific gamma variants could be assayed directly. The results indicate that mutations in many regions of gamma affect its binding to alpha beta. A mutant missing the last five carboxy-terminal residues (83-87) was totally lacking in inhibitory activity. However, it still bound to PDE alpha beta tightly, although with a 100-fold lower dissociation constant (approximately 5 nM) than that of wild-type gamma (approximately 50 pM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Derivatization of the catalytic subunits of the chloroplast ATPase by 2-azido-ATP and dicyclohexylcarbodiimide. Evidence for catalytically induced interchange of the subunits

Modifications of the catalytic beta subunits of the chloroplast ATPase (CF1-ATPase) are reported which support the proposal that all three subunits participate sequentially during catalysis. The beta subunits of the CF1-ATPase are sufficiently homogeneous to allow detection of their derivatization with dicyclohexylcarbodiimide (DCCD) or the substrate analog 2-azido-ATP by two-dimensional isoelectric focusing. Whether the DCCD reacts with the same beta subunit that tightly binds ATP has not been known. Our results show that when CF1-ATPase is covalently labeled with 2-azido-ATP followed by reaction with DCCD, different beta subunits are labeled. The DCCD labeling does not stop catalytic cooperativity of the CF1-ATPase and allows slow enzyme turnover. When the DCCD-modified enzyme catalyzes 2-azido-ATP cleavage and the enzyme with tightly bound nucleotide is photolyzed, both DCCD-modified and unmodified subunits are randomly labeled by the azido nucleotide. This result is as expected if during the catalytic cycle one beta subunit with unique properties is replaced by another subunit that gains these properties. The participation of all three subunits in the catalytic cycle is suggested by the apparent retention of catalytic cooperativity by the two remaining subunits after one subunit has already catalyzed 2-azido-ATP cleavage and been labeled.     

Visual transduction in rod outer segments

The visual transduction cascade of the retinal rod outer segment responds to light by decreasing membrane current. This ion channel is controlled by cyclic GMP which is, in turn, controlled by its synthesis and degradation by guanylate cyclase and phosphodiesterase, respectively. When light bleaches rhodopsin there is an induced exchange of GTP for GDP bound to the alpha subunit of the retinal G-protein, transducin (T). The T alpha.GTP then removes the inhibitory constraint of a small inhibitory subunit (PDE gamma) on the retinal cGMP phosphodiesterase (PDE). This results in activation of the PDE and in hydrolysis of cGMP. Recently both low and high affinity binding sites have been identified for PDE gamma on the PDE alpha/beta catalytic subunits. The discovery of two PDE gamma subunits, each with different binding affinities, suggests that a tightly regulated shut-off mechanism may be present.     

Modulation of retinal transducin and phosphodiesterase activities by synthetic peptides of the phosphodiesterase gamma-subunit

Synthetic peptides corresponding to various regions of the light-activated guanosine 3',5'-cyclic monophosphate phosphodiesterase (PDE) gamma-subunit (PDE gamma) from bovine retinal rod outer segments were synthesized and tested for their ability to inhibit PDE activity, and GTPase activity of transducin. One of these peptides, corresponding to PDE gamma residues 31-45, inhibited PDE activity and GTPase activity in a dose-dependent manner. The GTPase activity was inhibited by PDE gamma-3 non-competitively. This region of the PDE gamma subunit may be involved in the direct interaction of transducin and PDE alpha beta with PDE gamma.     

Regulation of retinal cGMP cascade by phosducin in bovine rod photoreceptor cells. Interaction of phosducin and transducin.

Photoexcitation of retinal rod photoreceptor cells involves the activation of cGMP enzyme cascade in which sequential activation of rhodopsin, transducin, and the cGMP phosphodiesterase in the rod outer segment constitutes the signal amplification mechanism. Phosducin, a 33-kDa phosphoprotein, has been shown to form a tight complex with the T beta gamma subunit of transducin. In this study, we examined the interaction of phosducin-T beta gamma and the possible regulatory role of phosducin on the cGMP cascade. Addition of phosducin to photolyzed rod outer segment (ROS) membrane reduced the GTP hydrolysis activity of transducin as well as the subsequent activation of the cGMP phosphodiesterase. Phosducin also inhibited the pertussis toxin-catalyzed ADP-ribosylation of transducin, indicating that the interaction between the T alpha and T beta gamma subunits of transducin was interrupted upon binding of phosducin. The inhibitory effects of phosducin were reversed by the addition of exogenous T beta gamma. These results suggest that phosducin is capable of regulating the amount of T beta gamma available to interact with T alpha to form the active transducin complex and thereby functions as a negative regulator of the cGMP cascade. The phosducin-induced alteration of the subunit organization of transducin was examined by chemical cross-linking method using para-phenyl dimaleimide as cross-linker. It was found that the cross-linking among T alpha and T beta gamma was blocked in the presence of phosducin. This result implies that T beta gamma may undergo a conformational change upon phosducin binding which leads to the release of T alpha. Since phosducin is a soluble protein, the interaction with transducin only occurs when transducin is dissociated from ROS disc membrane. Indeed, phosducin failed to dissociate membrane-bound transducin and did not inhibit the initial cycle of transducin activation as measured by the presteady state GTP hydrolysis. However, phosducin interacts effectively with transducin released into solution after the initial activation and blocks the re-binding of T alpha. T beta gamma to ROS membrane by forming a tight complex with T beta gamma. This interaction may play an important role in regulating the turnover of the cGMP cascade in photoreceptor cells.     

Transducin subunit stoichiometry and cellular distribution in rod outer segments

Transducin is a heterotrimeric GTP-binding protein found in the outer segment of vertebrate retinas that links the photoactivation of rhodopsin (R*) with activation of a robust type VI cGMP phosphodiesterase (PDE6). Association of the alpha subunit of Transducin (G(alphat)) with the beta-gamma complex (G(betagamma)) is necessary for interaction of the holoprotein with R* and exchange of a GTP for a previously bound GDP. We have investigated the abundances of the three Transducin subunits by eluting them from bovine rod outer segment membranes by centrifugation under various conditions in vitro. We find that a substantial amount of G(betagamma) is eluted from ROS under conditions that do not elute G(alphat) and that there is an overall three to fourfold molar excess of G(betagamma) to G(alphat) in rod outer segments. These results suggest that the production and/or turnover of G(alphat), G(beta), and G(gamma) in the rod outer segment are controlled independently.     

Regulation of retinal transducin by C-terminal peptides of rhodopsin.

Transducin is a multi-subunit guanine-nucleotide-binding protein that mediates signal coupling between rhodopsin and cyclic GMP phosphodiesterase in retinal rod outer segments. Whereas the T alpha subunit of transducin binds guanine nucleotides and is the activator of the phosphodiesterase, the T beta gamma subunit may function to link physically T alpha with photolysed rhodopsin. In order to determine the binding sites of rhodopsin to transducin, we have synthesized eight peptides (Rhod-1 etc.) that correspond to the C-terminal regions of rhodopsin and to several external and one internal loop region. These peptides were tested for their inhibition of restored GTPase activity of purified transducin reconstituted into depleted rod-outer-segment disc membranes. A marked inhibition of GTPase activity was observed when transducin was pre-incubated with peptides Rhod-1, Rhod-2 and Rhod-3. These peptides correspond to opsin amino acid residues 332-339, 324-331 and 317-321 respectively. Peptides corresponding to the three external loop regions or to the C-terminal residues 341-348 did not inhibit reconsituted GTPase activity. Likewise, Rhod-8, a peptide corresponding to an internal loop region of rhodopsin, did not inhibit GTPase activity. These findings support the concept that these specific regions of the C-terminus of rhodopsin serve as recognition sites for transducin.     

Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, was competent in nucleotide binding, and had normal N-terminal sequence. In F1 subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiments showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector.     

Thiol modification as a probe of conformational forms of the F1 ATPase of Escherichia coli and of the structural asymmetry of its beta subunits

The sulfhydryl groups of soluble and membrane-bound F1 adenosine triphosphatase of Escherichia coli were modified by reaction with the fluorescent thiol reagents 5-iodoacetamidofluorescein, 2-[(4'-iodoacetamido)anilino]naphthalene-6-sulfonic acid 4-[N-(iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenzo-2-oxa-1,3-d iaz ole and 2-[(4'-maleimidyl)anilino]naphthalene-6-sulfonic acid. Whereas gamma and delta subunits were always labeled by these reagents, the beta subunit reacted preferentially in the soluble enzyme, and the alpha subunit in the membrane-bound enzyme. This suggests that the soluble enzyme undergoes a conformational change on binding to the membrane. The three beta subunits of the soluble ATPase did not react with chemical reagents in a similar manner. One beta subunit was cross-linked to the epsilon subunit on treatment of the ATPase with 1-ethyl-3-[3-(dimethyl-amino)propyl]carbodiimide, as observed previously by L?tscher et al. [Biochemistry (1984) 23, 4134-4140]. A second beta subunit, which did not cross-link to the epsilon subunit, was modified preferentially by the fluorescent thiol reagents and by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole. The third beta subunit was less chemically reactive than the others. Both alpha and beta subunits of the soluble 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole-modified enzyme were labeled by the fluorescent thiol reagents. Thus, the modified enzyme, which is inactive, probably has a different conformation from the native soluble ATPase.