Mutation of the fourth cytoplasmic loop of rhodopsin affects binding of transducin and peptides derived from the carboxyl-terminal sequences of transducin alpha and gamma subunits

The role of the putative fourth cytoplasmic loop of rhodopsin in the binding and catalytic activation of the heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure the ability of G(t), or G(t)-derived peptides, to inhibit the photoregeneration of rhodopsin from its active metarhodopsin II state. We show that a peptide corresponding to residues 340-350 of the alpha subunit of G(t), or a cysteinyl-thioetherfarnesyl peptide corresponding to residues 50-71 of the gamma subunit of G(t), are able to interact with metarhodopsin II and inhibit its photoconversion to rhodopsin. Alteration of the amino acid sequence of either peptide, or removal of the farnesyl group from the gamma-derived peptide, prevents inhibition. Mutation of the amino-terminal region of the fourth cytoplasmic loop of rhodopsin affects interaction with G(t) (Marin, E. P., Krishna, A. G., Zvyaga T. A., Isele, J., Siebert, F., and Sakmar, T. P. (2000) J. Biol. Chem. 275, 1930-1936). Here, we provide evidence that this segment of rhodopsin interacts with the carboxyl-terminal peptide of the alpha subunit of G(t). We propose that the amino-terminal region of the fourth cytoplasmic loop of rhodopsin is part of the binding site for the carboxyl terminus of the alpha subunit of G(t) and plays a role in the regulation of betagamma subunit binding.     

Mechanism of action of monoclonal antibodies that block the light activation of the guanyl nucleotide-binding protein, transducin

Seven monoclonal antibodies to the alpha subunit (G alpha) of the frog photoreceptor guanyl nucleotide-binding protein (transducin or G-protein) have been characterized as to their effect on G-protein function, and this has been correlated in the accompanying paper (Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 262, 10839-10847) with the antibody-binding sites on G alpha tryptic fragments. Antibodies 4A, 7A, 7B, 7C, and 7D are members of a class of antibodies that block G-protein activation by light and therefore also block activation of the cGMP phosphodiesterase. All these blocking antibodies also block the interaction of G-protein with rhodopsin as measured by the light-scattering "binding signal," and as measured by the stabilization of meta-rhodopsin II by bound G-protein (extra-meta-rhodopsin II). The antibodies (or Fab fragments) also solubilize G alpha beta gamma from the membrane in the dark under isosmotic conditions and thus interfere with G alpha interaction with the membrane. Antibody 4A also blocks the extra-meta-rhodopsin II generated by G-protein-rhodopsin interaction in detergent solubilized membranes. Thus, even in the absence of phospholipids, antibody 4A blocks G-protein-rhodopsin interaction. Therefore, we suggest that the antibodies recognize a region of G alpha involved with binding to rhodopsin. An alternative hypothesis is that this antigenic site is a region of interaction between the alpha and beta gamma subunits, disruption of this interaction leading to removal of both the alpha and beta gamma subunits from the membrane and blocking interaction with rhodopsin. This does not seem to be the case because the antibodies immunoprecipitate the alpha beta gamma complex, and not just the alpha subunit. Other antibodies, 4C and 4H, do not block phosphodiesterase activation, the light-scattering signal, extra-meta-rhodopsin II formation, or interaction with the membrane in the dark and therefore recognize other sites on G alpha.     

Calcium channel beta subunits differentially regulate the inhibition of N-type channels by individual Gbeta isoforms

The direct inhibition of N- and P/Q-type calcium channels by G protein betagamma subunits is considered a key mechanism for regulating presynaptic calcium levels. We have recently reported that a number of features associated with this G protein inhibition are dependent on the G protein beta subunit isoform (Arnot, M. I., Stotz, S. C., Jarvis, S. E., Zamponi, G. W. (2000) J. Physiol. (Lond.) 527, 203-212; Cooper, C. B., Arnot, M. I., Feng, Z.-P., Jarvis, S. E., Hamid, J., Zamponi, G. W. (2000) J. Biol. Chem. 275, 40777-40781). Here, we have examined the abilities of different types of ancillary calcium channel beta subunits to modulate the inhibition of alpha(1B) N-type calcium channels by the five known different Gbeta subunit subtypes. Our data reveal that the degree of inhibition by a particular Gbeta subunit is strongly dependent on the specific calcium channel beta subunit, with N-type channels containing the beta(4) subunit being less susceptible to Gbetagamma-induced inhibition. The calcium channel beta(2a) subunit uniquely slows the kinetics of recovery from G protein inhibition, in addition to mediating a dramatic enhancement of the G protein-induced kinetic slowing. For Gbeta(3)-mediated inhibition, the latter effect is reduced following site-directed mutagenesis of two palmitoylation sites in the beta(2a) N-terminal region, suggesting that the unique membrane tethering of this subunit serves to modulate G protein inhibition of N-type calcium channels. Taken together, our data suggest that the nature of the calcium channel beta subunit present is an important determinant of G protein inhibition of N-type channels, thereby providing a possible mechanism by which the cellular/subcellular expression pattern of the four calcium channel beta subunits may regulate the G protein sensitivity of N-type channels expressed at different loci throughout the brain and possibly within a neuron.     

The E and G subunits of the yeast V-ATPase interact tightly and are both present at more than one copy per V1 complex

The E and G subunits of the yeast V-ATPase are believed to be part of the peripheral or stator stalk(s) responsible for physically and functionally linking the peripheral V1 sector, responsible for ATP hydrolysis, to the membrane V0 sector, containing the proton pore. The E and G subunits interact tightly and specifically, both on a far Western blot of yeast vacuolar proteins and in the yeast two-hybrid assay. Amino acids 13-79 of the E subunit are critical for the E-G two-hybrid interaction. Different tagged versions of the G subunit were expressed in a diploid cell, and affinity purification of cytosolic V1 sectors via a FLAG-tagged G subunit resulted in copurification of a Myc-tagged G subunit, implying more than one G subunit was present in each V1 complex. Similarly, hemagglutinin-tagged E subunit was able to affinity-purify V1 sectors containing an untagged version of the E subunit from heterozygous diploid cells, suggesting that more than one E subunit is present. Overexpression of the subunit G results in a destabilization of subunit E similar to that seen in the complete absence of subunit G (Tomashek, J. J., Graham, L. A., Hutchins, M. U., Stevens, T. H., and Klionsky, D. J. (1997) J. Biol. Chem. 272, 26787-26793). These results are consistent with recent models showing at least two peripheral stalks connecting the V1 and V0 sectors of the V-ATPase and would allow both stalks to be based on an EG dimer.     

The HA2 subunit of influenza hemagglutinin inserts into the target membrane prior to fusion

The interaction between influenza virus and target membrane lipids during membrane fusion was studied with hydrophobic photoactivatable probes. Two probes, the newly synthesized bisphospholipid diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine and the phospholipid analogue 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]-[2-3H]- undecanoyl]-sn-glycero-3-phosphocholine (Harter, C., B?chi, T., Semenza, G., and Brunner , J. (1988) Biochemistry 27, 1856-1864), were used. Both labeled the HA2 subunit of the virus at low pH. By measuring virus-liposome interactions at 0 degrees C, it could be demonstrated that HA2 was inserted into the target membrane prior to fusion. As we have recently demonstrated, at this temperature, exposure of the fusion peptide of HA2 takes place within 15 s after acidification, but fusion does not start for 4 min (Stegmann, T., White, J. M., and Helenius, A. (1990) EMBO J. 9, 4231-4241). HA2 was labeled at least 2 min before fusion. No labeling of the HA1 subunit was seen. These data indicate that fusion is triggered by a direct interaction of the HA2 subunit of a kinetic intermediate form of HA with the lipids of the target membrane. Most likely, it is the fusion peptide of HA2 that is inserted into the target membrane. Just before fusion, HA is thus an integral membrane protein in both membranes. In contrast, the bromelain-derived ectodomain of HA was labeled by 1-palmitoyl-2(11-[4-[3-(trifluoromethyl)diazirinyl]phenyl]- [2-3H]undecanoyl)-sn-glycerol-3-phosphocholine at low pH but not by diphosphatidylethanolamine trifluoromethyl [3H]phenyl diazirine. This indicates that insertion of the fusion peptide of the bromelain-derived ectodomain of HA into a membrane differs from that of viral HA during fusion.     

Two subunits of the canine signal peptidase complex are homologous to yeast SEC11 protein

Canine microsomal signal peptidase activity was previously isolated as a complex of five subunits (25, 22/23, 21, 18, and 12 kDa). Two of the signal peptidase complex (SPC) subunits (23/23 and 21 kDa) have been cloned and sequenced. One of these, the 21-kDa subunit, was observed to be a mammalian homolog of SEC11 protein (Sec11p) (Greenburg, G., Shelness, G. S., and Blobel, G. (1989) J. Biol. Chem. 264, 15762-15765) a gene product essential for signal peptide processing and cell growth in yeast (B?hni, P.C., Deshqies, R.J., and Schekman, R.W. (1988) J. Cell Biol. 106, 1035-1042). cDNA clones for the 18-kDa SPC subunit have now been characterized and found to encode a second SEC11p homolog. Both the 18- and 21-kDa canine SPC subunits are integral membrane proteins by virtue of their resistance to alkaline extraction. Upon detergent solubilization, both proteins are found in a complex with the 22/23 kDa SPC subunit, the only SPC subunit containing N-linked oligosaccharide. No steady-state pool of canine Sec11p-like monomers is detected in microsomal membranes. Alkaline extraction of microsomes prior to solubilization or solubilization at alkaline pH causes partial dissociation of the SPC. The Sec11p-like subunits displaced from the complex under these conditions demonstrate no signal peptide processing activity by themselves. The existence of homologous subunits is common to a number of known protein complexes and provides further evidence that the association between SPC proteins observed in vitro may be physiologically relevant to the mechanism of signal peptide processing and perhaps protein translocation.     

Evidence for direct interaction of Gs alpha with the Ca2+ channel of skeletal muscle.

The alpha subunits of heterotrimeric GTP-binding (G) proteins act upon ion channels through both cytoplasmic and membrane-delimited pathways (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213). The membrane pathway may involve either a direct interaction between G protein and ion channel or an indirect interaction involving a membrane-delimited second messenger. To distinguish between the two possibilities, we tested whether a purified G protein could interact with a purified channel protein in a defined system to produce changes in channel currents. We selected the alpha subunit of Gs and the dihydropyridine (DHP)-sensitive Ca2+ channel of skeletal muscle T-tubules, the DHP binding protein (DHPBP), because: 1) a membrane-delimited interaction between the two has been shown (Brown, A. M., and Birnbaumer, L. (1990) Annu. Rev. Physiol. 52, 197-213; Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895); and 2) at the present time, these Ca2+ channels are the only putative G protein channel effectors which, following purification, still retain channel function. We used a defined system in which purified components were studied by direct reconstitution in planar lipid bilayers. Just as we had found in crude skeletal muscle T-tubule membranes (Yatani, A., Imoto, Y., Codina, J., Hamilton, S. L., Brown, A. M., and Birnbaumer, L. (1988) J. Biol. Chem. 263, 9887-9895), alpha*s but not alpha*i-3 stimulated Ca2+ currents. However, in the reconstituted system, this probably represents a direct interaction between Gs alpha and Ca2+ channels. To establish whether the two proteins were physically associated in the native T-tubule membrane, we examined the ability of either endogenous G proteins or exogenous alpha*s to purify with detergent-solubilized DHPBP through a wheat germ agglutinin affinity column and a sucrose gradient. Small amounts of a labeled G protein were found to co-purify with DHPBP. In addition, partially purified DHPBP increased the sedimentation rate of purified alpha*s but not alpha*i-3. G proteins were immunoprecipitated with an antibody to the alpha 1 subunit of the DHPBP, and, in addition, both alpha s and the beta subunit of Gs were detected in Western blots of the partially purified DHPBP. The results suggest that Gs and Ca2+ channels are closely associated in the T-tubule plasma membrane, and we conclude that skeletal muscle Ca2+ channels are direct effectors for Gs.     

Immunogold localization of the regulatory subunit of a type II cAMP- dependent protein kinase tightly associated with mammalian sperm flagella

We have shown previously that the regulatory subunit (RII) of a type II cAMP-dependent protein kinase is an integral component of the mammalian sperm flagellum (Horowitz, J.A., H. Toeg, and G.A. Orr. 1984. J. Biol. Chem. 259:832-838; Horowitz, J.A., W. Wasco, M. Leiser, and G.A. Orr. 1988. J. Biol. Chem. 263:2098-2104). The subcellular localization of this flagellum-associated RII in bovine caudal epididymal sperm was analyzed at electron microscope resolution with gold-conjugated secondary antibody labeling techniques using anti-RII monoclonal antibodies. By immunoblot analysis, the flagellum-associated RII was shown to interact with mAb 622 which cross reacts with both neural and nonneural isoforms of RII. In contrast, a neural specific monoclonal antibody (mAb 526) failed to interact with flagellar RII. In the midpiece of the demembranated sperm tail, gold label after mAb 622 incubation was primarily associated with the outer mitochondrial membrane. Although almost all specific labeling in the midpiece can be assigned to the mitochondria, in the principal piece, there is some labeling of the fibrous sheath. Labeling of the outer dense fibers and the axoneme was sparse. Specific labeling was virtually absent in the sperm head. Sections of sperm tails incubated in the absence of primary antisera or with mAb 526 showed little labeling. A beta-tubulin monoclonal antibody localized only to the 9 + 2 axoneme. These results raise the possibility that a type II cAMP-dependent protein kinase located at the outer mitochondrial membrane plays a role in the direct cAMP stimulation of mitochondrial respiration during sperm activation.     

Functional expression of NADPH oxidase components (alpha- and beta-subunits of cytochrome b558 and 45-kDa flavoprotein) by intrinsic human glomerular mesangial cells.

Recently, we have shown that human glomerular mesangial cells (HMCs) release oxygen radicals from the plasma membrane in response to cytokines. Now we have used diphenylene iodonium, a covalent binding inhibitor of activated 45-kDa flavoprotein, in neutrophils radiolabeled with 125I and could identify a 45-kDa protein band in a separated HMC plasma membrane fraction. Low temperature difference spectroscopy showed a peak absorbance at 428 and 558 nm. Direct potentiometry of HMC membranes (-340 to -160 mV) showed the presence of a low potential cytochrome (76 pmol/mg to HMC membrane protein) identified as cytochrome b558. In slot blots, mouse monoclonal antibody (mAb) 7D5, specific for the extracellular domain of the alpha-subunit, showed a positive reaction with HMCs. In Western blots, mAb 449, directed against the cytoplasmic epitope of the alpha-subunit, identified a 23-kDa protein; and mAb 48, raised against the large (beta) subunit of cytochrome b558 of human neutrophils (Verhoeven, A. J., Bolscher, B. G. J. M., Meerhof, L. J., van Zwieten, R., Keijer, J., Weening, R. S., and Roos, D. (1989) Blood 73, 1686-1694), detected a smear between 75 and 100 kDa in denatured HMC membrane protein. These data determined with HMCs, suggest for the first time the expression of three essential components of NADPH:O2- oxidoreductase in mesenchymal cells.     

Structural requirements for the stabilization of metarhodopsin II by the C terminus of the alpha subunit of transducin

The retinal receptor rhodopsin undergoes a conformational change upon light excitation to form metarhodopsin II (Meta II), which allows interaction and activation of its cognate G protein, transducin (G(t)). A C-terminal 11-amino acid peptide from transducin, G(talpha)-(340-350), has been shown to both bind and stabilize the Meta II conformation, mimicking heterotrimeric G(t). Using a combinatorial library we identified analogs of G(talpha)-(340-350) that bound light-activated rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). We have made peptides with key substitutions either on the background of the native G(talpha)-(340-350) sequence or on the high affinity sequences and used the stabilization of Meta II as a tool to determine which amino acids are critical in G protein-rhodopsin interaction. Removal of the positive charge at the N termini by acylation or delocalization of the charge by K to R substitution enhances the affinity of the G(talpha)-(340-350) peptides for Meta II, whereas a decrease was observed following C-terminal amidation. Cys-347, a residue conserved in pertussis toxin-sensitive G proteins, was shown to interact with a hydrophobic site in Meta II. These studies provide further insight into the mechanism of interaction between the G(talpha) C terminus and light-activated rhodopsin.     

Integration of F1 and the membrane sector of the proton-ATPase of Escherichia coli. Role of subunit "b" (uncF protein)

Membranes derived from the Escherichia coli strain AN1460 which carries the multicopy plasmid pAN45 (unc+) (Downie, J. A., Langman, L., Cox, G. B., Yanofsky, C., and Gibson, (1980) J. Bacteriol. 143, 8-17) were enriched 5- to 10-fold in proton-ATPase activity. Incubation of F1-depleted AN1460 membranes with trypsin abolished F1-binding ability but did not inhibit proton transport through the membrane sector (F0). Sodium dodecyl sulfate-gel electrophoresis indicated that subunit "b" (uncF protein) of F0 was cleaved by trypsin and prebound F1 protected against the trypsin effect. Subunits "a" (uncB protein) and "c" (uncE protein) were unaffected by the trypsin treatment. A water-soluble fragment (Mr = 14,800) was liberated after trypsin treatment and appeared to arise from subunit b. Studies of enzyme hybridization and of F1 binding to membranes derived from strains containing mutations in uncB, F, and E genes supported the suggestion that subunit b is involved in F1 binding to the F0. Also, extraction of membranes with KSCN increased the relative proportion of subunit b in the membrane and this coincided with a parallel increase in trypsin-sensitive F1-binding ability. It is proposed that subunit b is involved in binding of F1 to the F0; this agrees with the presumed role of the protein as deduced from predictions of its secondary and tertiary structure (Walker, J. E., Saraste, M., and Gay, N. J. (1982) Nature (Lond.) 298, 867-869; Senior, A. E. (1983) Biochim. Biophys. Acta, in press).     

Subunit stoichiometry of the chloroplast photosystem II antenna system and aggregation state of the component chlorophyll a/b binding proteins

Photosystem (PS) II membranes, obtained by the method of Berthold et al. (Berthold, D. A., Babcock, G. T., and Yocum, C. F. (1981) FEBS Lett. 134, 231-234), have been fractionated by a sucrose gradient ultracentrifugation method which allows the quantitative separation of the three major chlorophyll binding complexes in these membranes: the chlorophyll (chl) a binding PSII reaction center core, the major light-harvesting complex II, and the minor chl a/b proteins called CP26, CP29, and CP24. Each fraction has been analyzed for its subunit stoichiometry by quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis methods. The results show that 12 mol of light-harvesting complex II and 1.5 mol of each of the minor chl a/b proteins are present per mol of the PSII reaction center complex in PSII membranes. These data suggest a dimeric organization of PSII, in agreement with a recent crystallographic study (Bassi, R., Ghiretti Magaldi, A., Tognon, G., Giacometti, G. M., and Miller, K. (1989) Eur. J. Cell Biol. 50, 84-93) and imply that such a dimeric complex is served by antenna chl a/b proteins whose minimal aggregation state includes three polypeptides. This was confirmed by covalent cross-linking of purified antenna complexes.     

RACK1 binds to a signal transfer region of G betagamma and inhibits phospholipase C beta2 activation

Receptor for Activated C Kinase 1 (RACK1), a novel G betagamma-interacting protein, selectively inhibits the activation of a subclass of G betagamma effectors such as phospholipase C beta2 (PLCbeta2) and adenylyl cyclase II by direct binding to G betagamma (Chen, S., Dell, E. J., Lin, F., Sai, J., and Hamm, H. E. (2004) J. Biol. Chem. 279, 17861-17868). Here we have mapped the RACK1 binding sites on G betagamma. We found that RACK1 interacts with several different G betagamma isoforms, including G beta1gamma1, Gbeta1gamma2, and Gbeta5gamma2, with similar affinities, suggesting that the conserved residues between G beta1 and G beta5 may be involved in their binding to RACK1. We have confirmed this hypothesis and shown that several synthetic peptides corresponding to the conserved residues can inhibit the RACK1/G betagamma interaction as monitored by fluorescence spectroscopy. Interestingly, these peptides are located at one side of G beta1 and have little overlap with the G alpha subunit binding interface. Additional experiments indicate that the G betagamma contact residues for RACK1, in particular the positively charged amino acids within residues 44-54 of G beta1, are also involved in the interaction with PLCbeta2 and play a critical role in G betagamma-mediated PLCbeta2 activation. These data thus demonstrate that RACK1 can regulate the activity of a G betagamma effector by competing for its binding to the signal transfer region of G betagamma.     

Dissociation, cross-linking, and glycosylation of the coated vesicle proton pump

In order to refine further our structural model of the coated vesicle (H+)-ATPase (Arai, H., Terres, G., Pink, S., and Forgac, M. (1988) J. Biol. Chem. 263, 8796-8802), we have extended our structural analysis to identify peripheral and glycosylated subunits of the pump as well as to identify subunits which are in close proximity in the native (H+)-ATPase complex. Treatment of the purified, reconstituted (H+)-ATPase with 0.30 M KI in the presence or absence of ATP or MgATP results in the release of the 73-, 58-, 40-, 34-, and 33-kDa subunits, leaving behind the 100-, 38-, 19-, and 17-kDa subunits in the membrane. Because the former group of polypeptides is released from the membrane in the absence of detergent, they correspond to peripheral membrane proteins. To determine which subunits are in close proximity, cross-linking of the purified (H+)-ATPase was carried out using the cleavable, bifunctional amino reagent 3,3'-dithiobis(sulfosuccinimidylpropionate) followed by two-dimensional gel electrophoresis. These studies indicate that contact regions exist between the 73- and 58-kDa subunits as well as between the 17-kDa subunit and the 40-, 34-, and 33-kDa subunits. To test for glycosylation of the (H+)-ATPase, the detergent-solubilized complex was treated with neuraminidase followed by electrophoresis and blotting using a peanut lectin/horseradish peroxidase conjugate. Galactose-inhibitable staining of the 100-kDa subunit, together with affinity chromatography of the intact (H+)-ATPase on peanut lectin agarose, indicates that the 100-kDa subunit is glycosylated, most likely at a site exposed on the luminal side of the membrane. These results, together with those presented in the preceding paper (Adachi, I., Arai, H., Pimental, R., and Forgac, M. (1990) J. Biol. Chem. 265, 960-966), were used in the construction of a refined model of the coated vesicle (H+)-ATPase.     

A role for membrane potential in the biogenesis of cytochrome c oxidase subunit II, a mitochondrial gene product

Subunit II of yeast cytochrome c oxidase is synthesized on mitochondrial ribosomes as a precursor containing a transient NH2-terminal presequence and is inserted into the mitochondrial inner membrane from the matrix side. Using an optimized in vitro mitochondrial translation system (McKee, E.E., and Poyton, R. O. (1984) J. Biol. Chem. 259, 9320-9331), we have examined the requirement for an electrochemical potential (delta mu H+) across the inner mitochondrial membrane during subunit II biogenesis. When mitochondrial gene products are synthesized under conditions that prevent formation of a normal delta mu H+, accumulation of unprocessed subunit II (pre-II) occurs. The majority of pre-II generated in this way is inserted into the lipid bilayer, as judged by resistance to extraction with 0.1 M Na2CO3. Therefore, it appears that a delta mu H+ is required for the normal biogenesis of subunit II, and that the delta mu H+ is required for a function other than the insertion of pre-II into the lipid bilayer of the inner membrane.     

Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.     

Spectroscopic evidence for an intermediate in the T6 to R6 allosteric transition of the Co(II)-substituted insulin hexamer.

The phenol-induced conformational transition in the insulin hexamer is known to involve a large change in structure wherein residues 1-8 of the insulin B-chain are transformed from an extended coil (T-state) to a helix (R-state). This change in protein conformation both exposes a cryptic protein pocket on each subunit to which phenol binds and forces the HisB10 zinc sites to undergo a change in coordination geometry from octahedral to tetrahedral [Derewenda, U., Derewenda, Z., Dodson, E. J., Dodson, G. G., Reynolds, C. D., Smith, G. D., Sparks, C., & Swensen, D. (1989) Nature 338, 593-596]. Substitution of Co(II) for Zn(II) at the HisB10 sites introduces a sensitive chromophoric probe of the structural and chemical events that occur during this allosteric transition [Roy, M., Brader, M. L., Lee, R. W.-K., Kaarsholm, N. C., Hansen, J. F., & Dunn, M. F. (1989) J. Biol. Chem. 264, 19081-19085]. In this study, using rapid-scannig stopped-flow (RSSF) UV-visible spectroscopic studies, we demonstrate that a transient chemical intermediate is formed during the phenol-induced conversion of Co(II)-substituted hexamer from the T-state to the R-state. Decomposition of the RSSF spectra gave a spectrum for the intermediate with d-d transitions consistent with the assignment of the intermediate as either a distorted tetrahedral or a 5-coordinate Co(II) species. Possible structures for the intermediate and the implications of these findings to the allosteric mechanism are considered.     

Signal peptidases recognize a structural feature at the cleavage site of secretory proteins

The cloning of the gene for staphylococcal nuclease A in the pIN-III-OmpA secretion vector results in a hybrid protein which is processed by signal peptidase I, yielding an active form of the nuclease that is secreted across the cytoplasmic membrane (Takahara, M., Hibler, D., Barr, P. J., Gerlt, J. A., and Inouye, M. (1985) J. Biol. Chem. 260, 2670-2674). Using oligonucleotide-directed site-specific mutagenesis, we have constructed a set of mutants at the cleavage site area of the precursor hybrid protein designed to alter progressively the predicted secondary structure of the cleavage site. Our results show that processing becomes increasingly defective as the turn probability decreases. These results are consistent with the structural requirement that we found for the processing of lipoprotein by signal peptidase II (Inouye, S., Duffaud, G., and Inouye, M. (1986) J. Biol. Chem. 261, 10970-10975). We conclude that secretory precursor proteins have a distinct secondary structural requirement at their cleavage site for processing by signal peptidase I, as well as by signal peptidase II.     

Structural and functional studies of cross-linked Go protein subunits

The guanine nucleotide binding proteins (G proteins) that couple hormone and other receptors to a variety of intracellular effector enzymes and ion channels are heterotrimers of alpha, beta, and gamma subunits. One way to study the interfaces between subunits is to analyze the consequences of chemically cross-linking them. We have used 1,6-bismaleimidohexane (BMH), a homobifunctional cross-linking reagent that reacts with sulfhydryl groups, to cross-link alpha to beta subunits of Go and Gi-1. Two cross-linked products are formed from each G protein with apparent molecular masses of 140 and 122 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both bands formed from Go reacted with anti-alpha o and anti-beta antibody. The mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is anomalous since the undenatured, cross-linked proteins have the same Stokes radius as the native, uncross-linked alpha beta gamma heterotrimer. Therefore, each cross-linked product contains one alpha and one beta subunit. Activation of Go by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) does not prevent cross-linking of alpha to beta gamma, consistent with an equilibrium between associated and dissociated subunits even in the presence of GTP gamma S. The same cross-linked products of Go are formed in brain membranes reacted with BMH as are formed in solution, indicating that the residues cross-linked by BMH in the pure protein are accessible when Go is membrane bound. Analysis of tryptic peptides formed from the cross-linked products indicates that the alpha subunit is cross-linked to the 26-kDa carboxyl-terminal portion of the beta subunit. The cross-linked G protein is functional, and its alpha subunit can change conformation upon binding GTP gamma S. GTP gamma S stabilizes alpha o to digestion by trypsin (Winslow, J.W., Van Amsterdam, J.R., and Neer, E.J. (1986) J. Biol. Chem. 261, 7571-7579) and also stabilizes the alpha subunit in the cross-linked product. Cross-linked G o can be ADP-ribosylated by pertussis toxin. This ADP-ribosylation is inhibited by GTP gamma S with a concentration dependence that is indistinguishable from that of the control, uncross-linked G o. These two kinds of experiments indicate that alpha o is able to change its conformation even though it cannot separate completely from beta gamma. Thus, although dissociation of the subunits accompanies activation of G o in solution, it is not obligatory for a conformational change to occur in the alpha subunit.