Molecular organization and pigment composition of R-phycoerythrin from the red alga Callithamnion rubosum

p3phycoerythrin is the major phycobiliprotein of Rhodophyta and endows these algae with the characteristic color. R-phycoerythrin purified from red alga Calithamnion rubosom is composed of four dissimilar polypeptide subunits, alpha, beta, gamma, and delta. In calibrated SDS gel electrophoresis their molecular weights are 21 000, 21 600, 31 000 and 33 000 daltons, respectively. The stoichiometry of the subunits in the native protein is 9 alpha: 9 beta: 2 gamma: 1 delta. R-phycoerythrin carries two covalently linked apoprotein red tetrapyrrol pigments: phycoerythrobilin (PEB) and phycourobilin (PUB). Chemical and spectroscopic data show that alpha subunit carries solely two PEB chromophores, beta subunit--3 PEB and 1 PUB groups, gamma subunit--3 PEB and 2 PUB groups and delta subunit--1 or 2 PEB and 1 PUB groups. The chromophore and polypeptide structure of R-phycoerythrin is mostly composed of all known phycobiliproteins of red and blue-green algae.     

Bilin attachment sites in the alpha, beta, and gamma subunits of R-phycoerythrin. Structural studies on singly and doubly linked phycourobilins

Three unique bilin peptides, a beta subunit peptide bearing a doubly linked phycourobilin (PUB), and two gamma subunit peptides with singly linked PUB groups, were obtained by enzymatic degradation of Gastroclonium coulteri R-phycoerythrin. These peptides were shown to have the sequences (Klotz, A. V., and Glazer, A. N. (1985) J. Biol. Chem. 260, 4856-4863): (Formula: see text) The sequence of peptide beta-3T was identical to that previously established for a doubly linked phycoerythrobilin (PEB) peptide derived from a B-phycoerythrin (Lundell, D. J., Glazer, A. N., DeLange, R. J., and Brown, D. M. (1984) J. Biol. Chem. 259, 5472-5480). Secondary ion mass spectrometry of beta-3T yielded a protonated molecular ion of 1629 mass units, the same as that given by the doubly linked PEB peptide (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5481-5484), indicating that the doubly linked PUB and PEB tetrapyrroles were isomeric structures. High resolution 1H NMR analyses of peptides beta-3T, gamma-BV8, and gamma-DP provided unambiguous structural assignments for the singly and doubly linked PUB chromophores and indicated that the peptides in gamma-BV8 and gamma-DP were linked to ring A. The determination of which peptide fragment is linked to ring A and which to ring D in peptide beta-3T was not achieved in this study. 1H NMR analyses of three PEB-peptides from G. coulteri R-phycoerythrin--alpha-1 Cys(PEB)-Tyr-Arg, alpha-2 Leu-Cys(PEB)-Val-Pro-Arg, and beta-1 Met-Ala-Ala-Cys(PEB)-Leu-Arg--showed that they were identical to previously described corresponding chromopeptides from Porphyridium cruentum B-phycoerythrin, with the peptide linked to ring A of PEB in each instance (Schoenleber, R. W., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5485-5489). This is the first documented report on the structure of singly or doubly linked phycourobilins.     

Phycoerythrins of marine unicellular cyanobacteria. II. Characterization of phycobiliproteins with unusually high phycourobilin content

A survey of marine unicellular cyanobacterial strains for phycobiliproteins with high phycourobilin (PUB) content led to a detailed investigation of Synechocystis sp. WH8501. The phycobiliproteins of this strain were purified and characterized with respect to their bilin composition and attachment sites. Amino-terminal sequences were determined for the alpha and beta subunits of the phycocyanin and the major and minor phycoerythrins. The amino acid sequences around the attachment sites of all bilin prosthetic groups of the phycocyanin and of the minor phycoerythrin were also determined. The phycocyanin from this strain carries a single PUB on the alpha subunit and two phycocyanobilins on the beta subunit. It is the only phycocyanin known to carry a PUB chromophore. The native protein, isolated in the (alpha beta)2 aggregation state, displays absorption maxima at 490 and 592 nm. Excitation at 470 nm, absorbed almost exclusively by PUB, leads to emission at 644 nm from phycocyanobilin. The major and minor phycoerythrins from strain WH8501 each carry five bilins per alpha beta unit, four PUBs and one phycoerythrobilin. Spectroscopic properties determine that the PUB groups function as energy donors to the sole phycoerythrobilin. Analysis of the bilin peptides unambiguously identifies the phycoerythrobilin at position beta-82 (residue numbering assigned by homology with B-phycoerythrin; Sidler, W., Kumpf, B., Suter, F., Klotz, A. V., Glazer, A. N., and Zuber, H. (1989) Biol. Chem. Hoppe-Seyler 370, 115-124) as the terminal energy acceptor in phycoerythrins.     

Phycoerythrins of marine unicellular cyanobacteria. I. Bilin types and locations and energy transfer pathways in Synechococcus spp. phycoerythrins

Marine Synechococcus strains WH8103, WH8020, and WH7803 each possess two different phycoerythrins, PE(II) and PE(I), in a weight ratio of 2-4:1. PE(II) and PE(I) differ in amino acid sequence and in bilin composition and content. Studies with strain WH7803 indicated that both PE(II) and PE(I) were present in the same phycobilisome rod substructures and that energy absorbed by PE(II) was transferred to PE(I). Strain WH8103 and WH8020 PE(I)s carried five bilin chromophores thioether-linked to cysteine residues in sequences homologous to those previously characterized in C-, B-, and R-PEs. In contrast, six bilins were attached to strain WH8103 and WH8020 PE(II)s. Five of these were at positions homologous to bilin attachment sites in other phycoerythrins. The additional bilin attachment site was on the alpha subunit. The locations and bilin types in these PE(s) and in the marine Synechocystis strain WH8501 PE(I) (Swanson, R. V., Ong, L. J., Wilbanks, S. M., and Glazer, A. N. (1991) J. Biol. Chem. 266, 9528-9534) are: (table; see text) Since phycourobilin (PUB) (lambda max approximately 495 nm) transfers energy to phycoerythrobilin (PEB) (lambda max approximately 550 nm), inspection of these data shows that the invariant PEB group at beta-82 is the terminal energy acceptor in phycoerythrins. The adaptations to blue-green light, high PUB content and the presence of an additional bilin on the alpha subunit, increase the efficiency of light absorption by PE(II)s at approximately 500 nm.     

Biliprotein light-harvesting strategies, phycoerythrin 566

A series of experiments on the light-harvesting properties of the cryptomonad biliprotein phycoerythrin 566 has been carried out on purified protein isolated from Cryptomonas ovata. Although this pigment has an absorption maximum at 566 nm, a property very close to that of other phycoerythrins, it was found to have a totally unique set of chromophores. The chromophores (bilins) responsible for its absorption spectrum were analyzed by a number of approaches. Chromophore-containing peptides were produced by trypsin treatment and purified in order to isolate the individual peptide-bound bilins free of overlapping absorption. These chromopeptides, after comparison with appropriate controls, showed that three spectrally distinct bilins occurred on the purified oligomeric protein. Two of the bilins were the well-known phycoerythrobilin and cryptoviolin, but the third was previously undiscovered and had an absorption spectrum between that of cryptoviolin and phycocyanobilin. Since the spectral diversity of the three bilins was fully maintained in solvents that minimize the effects of apoprotein on the spectra of the bilins, it is likely that the three bilins are also structurally dissimilar. The alpha and beta subunits, which constitute the protein, were separated by ion-exchange chromatography, and the new bilin was found to be the sole chromophore on the alpha subunit. It was also found that at least two alpha subunits could be separated and they both had this unusual bilin (cryptobilin 596). The beta subunit, therefore, contained both phycoerythrobilin and cryptoviolin. On the basis of the spectra of the three chromopeptides, the absorption spectrum of the protein was modeled using the known absorptivities of cryptoviolin and phycoerythrobilin.     

Subunit location and sequences of the cysteinyl peptides of pig heart NAD-dependent isocitrate dehydrogenase

Pig heart NAD-dependent isocitrate dehydrogenase has a subunit structure consisting of alpha 2 beta gamma, with the alpha subunit exhibiting a molecular weight of 39,000 and the beta and gamma each having molecular weights of 41,000. The amino-terminal sequences (33-35 residues) and the cysteinyl peptide sequences have now been determined by using subunits separated by chromatofocusing or isoelectric focusing and electroblotting. Displacement of the N-terminal sequence of the alpha subunit by 11-12 amino acids relative to that of the larger beta and gamma subunits reveals a 17 amino acid region of great similarity in which 10 residues are identical in all three subunits. The complete enzyme has 6.0 free SH groups per average subunit of 40,000 daltons, but yields 15 distinguishable cysteines in isolated tryptic peptides. Six distinct cysteines in sequenced peptides have been located in the alpha subunit. The beta and gamma subunits contain seven and five cysteines, respectively, with tryptic peptides containing three cysteines being common to the beta and gamma subunits. The three subunits appear to be closely related, but beta and gamma are more similar to each other than either is to the alpha subunit. The NAD-specific isocitrate dehydrogenase from pig heart has been shown to have 2 binding sites/enzyme tetramer for isocitrate, manganous ion, NAD+, and the allosteric activator ADP [Colman, R. F. (1983) Pept. Protein Rev. 1, 41-69]. It is proposed that the catalytically active tetrameric enzyme is organized as a dimer of dimers in which the alpha beta and alpha gamma dimers are nonidentical but functionally similar.     

Phycobiliprotein-bilin linkage diversity. II. Structural studies on A- and D-ring-linked phycoerythrobilins

The discovery that the phycocyanobilin group attached to Cys-155 of the beta subunit of C-phycocyanin is D-ring linked (Bishop, J. E., Lagarias, J. C., Nagy, J. O., Schoenleber, R. W., Rapoport, H., Klotz, A. V., and Glazer, A. N. (1986) J. Biol. Chem. 261, 6790-6796) prompted examination of the linkage mode for phycoerythrobilin (PEB) groups attached at the corresponding position in other biliproteins. Appropriate small peptides were obtained by exhaustive enzymatic digestion of Porphyridium cruentum R-phycocyanin (peptide R-PC beta-2TP PEB) and B-phycoerythrin (peptide B-PE beta-2TP PEB). These peptides had the following structures R-PC beta-2TP PEB Gly-Asp-Cys(PEB)-Ser-Ser B-PE beta-2TP PEB Cys(PEB)-Thr-Ser. The spectroscopic and chemical properties of these peptides were compared with those of P. cruentum B-phycoerythrin peptide alpha-1 PEB, Cys(PEB)-Tyr-Arg, in which the bilin is A-ring linked (Schoenleber, R. W., Leung, S.-L., Lundell, D. J., Glazer, A. N., and Rapoport, H. (1983) J. Am. Chem. Soc. 105, 4072-4076). The PEB groups in peptides R-PC beta-2TP PEB and B-PE beta-2TP PEB were shown to be D-ring linked on the basis of the following criteria. Secondary ion mass spectrometry showed the bilins in these peptides and in alpha-1 PEB to have the same mass. The 18'-CH3, 18'-H, and 15-H resonances in the 1H NMR spectra of R-PC beta-2TP PEB and B-PE beta-2TP PEB appear significantly upfield from the corresponding thioether-linked ring A resonances seen in the spectrum of peptide alpha-1 PEB. The CD spectra of the two former peptides showed a strong positive Cotton effect at 300 nm. Such a Cotton effect is absent from the CD spectrum of peptide alpha-1 PEB and those of other A-ring-linked PEB peptides. Refluxing in methanol led to a near-quantitative release of PEB from alpha-1 PEB but no release from R-PC beta-2TP PEB and less than 20% release from B-PE beta-2TP PEB. In conjunction with earlier studies, these results show that distinctive amino acid sequences are found about the attachment sites for A-ring-linked, D-ring-linked, and dilinked (A- and D-ring-linked) bilins on the alpha and beta subunits of cyanobacterial and red algal phycobiliproteins and that the mode of linkage can be correctly predicted from inspection of the amino acid sequence.     

Isolation and partial characterization of two different subunits from the molybdenum-iron protein of Azotobacter vinelandii nitrogenase

The molybdenum-iron protein of Azotobacter vinelandii nitrogenase was separated into two subunits of equal concentration by ion exchange chromatography on sulfopropyl (SP) Sephadex at pH 5.4 in 7 M urea. Better than 90% yield of each subunit was obtained on a preparative scale if the reduced carboxymethylated molybdenum-iron protein was incubated at 45 degrees C for 45 min prior to chromatography. Without the heating step low yields of the subunits were obtained. Although the amino acid compositions of the two subunits were very similar, the NH2-terminal sequences were completely different as determined by automated sequential Edman degradation. The sequence for the alpha subunit was NH2-Ser-Gln-Gln-Val-Asp-Lys-Ile-Lys-Ala-Ser-Tyr-Pro-Leu-Phe-Leu-Asp-Gln-Asp-Tyr- and for the beta subunit the sequence was NH2-Thr-Gly-Met-Ser-Arg-Glu-Glu-Val-Glu-Ser-Leu-Ile-Gln-Glu-Val-Leu-Glu-Val-Tyr-. Likewise the COOH-terminal sequences for the two subunits, as determined with carboxypeptidase Y, were tota-ly different. The sequence for the alpha subunit was -Leu-Arg-Val-COOH and that for the beta subunit was -Ile-(Phe, Glu)-Ala-Phe-COOH. Radioautographs of tryptic peptide maps were prepared for the molybdenum-iron protein and the two subunits which had been labeled at the cysteinyl residues with iodo[2-14C]acetic acid. These maps indicated that the two subunits had no cysteinyl peptides in common and that the cysteinyl residues were clustered in both subunits.     

In vitro attachment of bilins to apophycocyanin. III. Properties of the phycoerythrobilin adduct

Addition of phycoerythrobilin (PEB) to apophycocyanin at pH 7.0 resulted in covalent adduct formation. The adduct showed absorbance maxima at 575 and 605 nm and fluorescence emission maxima at 582 and 619 nm. Analysis of bilin peptides obtained upon tryptic digestion of the adduct showed residues alpha-Cys-84 and beta-Cys-82 to be the sites of bilin addition. The product of PEB addition at the alpha-Cys-84 site was shown by 1H NMR analysis to be a dihydrobiliviolinoid peptide-linked pigment differing in structure from that of the naturally occurring PEB-adduct by the presence of a double bond in between C2 and C3 of ring A. At the beta-Cys-82 site both a dihydrobiliviolinoid and a PEB adduct were obtained. Biliverdin also formed a covalent adduct with apophycocyanin with a lambda max of 669 nm. These results show that the spontaneous in vitro addition of bilins to apophycocyanin does not exhibit the site selectivity of bilin addition observed in vivo. This offers the opportunity to form novel semisynthetic phycobiliproteins.     

In vitro attachment of bilins to apophycocyanin. I. Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C-phycocyanin

Expression of cloned alpha and beta subunit genes of Synechococcus sp. PCC7002 C-phycocyanin in Escherichia coli led to the production of large amounts of apophycocyanin. The apophycocyanin was purified to homogeneity and shown to be an alpha beta monomer. The reactivity of the apoprotein toward a number of open chain and cyclic tetrapyrroles was examined. Phycocyanobilin (PCB), phycoerythrobilin, and biliverdin all formed covalent adducts with apophycocyanin in 50 mM sodium phosphate buffer at pH 7.0. Mesobiliverdin, bilirubin, PCB dimethyl ester, protoporphyrin IX, and hemin did not react with the apoprotein. None of these tetrapyrroles reacted with 2 mM 2-mercaptoethanol, cysteine, or reduced glutathione under the same conditions. The adduct with PCB was investigated in greater detail. Its visible absorption spectrum, with a maximum at 646 nm, is more similar to that of allophycocyanin than phycocyanin. Two PCBs are bound per alpha beta monomer when the reaction is performed with excess bilin. While tryptic digestion of the adduct generates numerous bilin peptides, amino acid analysis of these chromopeptides revealed that PCB reacted specifically at alpha-Cys-84 and beta-Cys-82, two of the three cysteinyl residues that serve as the attachment sites for PCB in native phycocyanin. The major bilin peptides arising from in vitro adduct formation at each of these sites differed both in chromatographic behavior and in spectroscopic properties from the corresponding PCB peptides isolated from tryptic digests of native C-phycocyanin.     

Phycobilins of cryptophycean algae. Occurrence of dihydrobiliverdin and mesobiliverdin in cryptomonad biliproteins.

Structures of the open-chain tetrapyrrole (bilin) prosthetic groups of the cryptophycean biliproteins phycocyanin 645 (Cr-PC 645; from strain UW374), phycoerythrin 566 (Cr-PE 566; from strain Bermani) and phycoerythrin 545 (Cr-PE 545; from Proteomonas sulcata Hill & Wetherbee) were examined by absorption, 1H NMR spectroscopy, and mass spectrometry. These biliproteins carry the following covalently attached bilins: Cr-PC 645 (alpha subunit) has one mesobiliverdin, (beta subunit), two phycocyanobilins and a doubly linked 15,16-dihydrobiliverdin; Cr-PC 566 (alpha), bilin 584, (beta), phycoerythrobilin and two bilin 584 chromophores (Wedemayer, G.J., Wemmer, D.E., and Glazer, A.N. (1991) J. Biol. Chem. 266, 4731-4741); Cr-PE 545 (alpha) has one 15,16-dihydrobiliverdin and (beta), only phycoerythrobilins. This is the first report of naturally occurring biliproteins carrying either 15,16-dihydrobiliverdin or mesobiliverdin chromophores. Native cryptomonad phycobiliproteins have been classified on the basis of the position of their long wavelength absorption maxima. However, comparison of the bilins of Cr-PE 566 from strain Bermani with those of Cr-PE 566 of strain CBD shows that the two proteins carry different bilins on the alpha subunit. Consequently, the identity of the bilin prosthetic groups on cryptophycean phycobiliproteins cannot be unambiguously inferred from simple inspection of the visible absorption spectra.     

The suicide inactivation of ox liver short-chain acyl-CoA dehydrogenase by propionyl-CoA. Formation of an FAD adduct.

R-Phycoerythrin contains two covalently bound bilin prosthetic groups, phycoerythrobilin and phycourobilin. The two chromophore types were separated as their peptide-bound derivatives by subjecting tryptic digests of R-phycoerythrin to adsorption chromatography on Sephadex G-25. The structure and apoprotein linkages of the bound phycoerythrobilin were found to be identical with those previously reported for this phycobilin [Killilea, O'Carra & Murphy (1980) Biochem. J. 187, 311-320]. Phycourobilin is a tetrapyrrole, containing no oxo bridges and has the same order of side chains as IX alpha bilins. The chromophore is linked to the peptide through two and possibly three of its pyrrole rings. One linkage possibly consists of an ester bond between the hydroxy group of a serine residue and the propionic acid side chain of one of the inner rings. The second linkage is a labile thioether bond between a cysteine residue and the C2 side chain of pyrrole ring A. The third linkage is a stable thioether bond between a cysteine residue and the alpha-carbon atom of the C2 side chain of pyrrole ring D. Ring D is unsaturated and is attached to ring C through a saturated carbon bridge. Rings B and C have a conjugated system of five bonds, as found in other urobilinoid pigments. Ring A is attached to ring B via a saturated carbon bridge. Both of the alpha-positions of ring A are in the reduced state, but the ring does contain an unsaturated centre (probably a double bond between the beta-carbon and the ring nitrogen atom). The presence of this double bond and its isomerization into the bridge position between rings A and B would explain the extension of the conjugated system of phycourobilin to that of a phycoerythrobilinoid/rhodenoid pigment in acid or alkali.     

Phycobilins of cryptophycean algae. Structures of novel bilins with acryloyl substituents from phycoerythrin 566

Cryptomonad strain CBD phycoerythrin 566 carries four open-chain tetrapyrrole (bilin) prosthetic groups: three singly thioether-linked bilins at alpha-19, beta-82, and beta-158 and a bilin linked through two thioether bonds at beta-50,61 (amino acid sequence numbering from Wilbanks, S. M., Wedemayer, G.J., and Glazer, A.N. (1989) J. Biol. Chem. 264, 17860-17867). The structures of all four peptide-linked prosthetic groups were determined by 1H NMR spectroscopy. The bilin at beta-82 was identified as phycoerythrobilin (PEB), a common prosthetic group in cyanobacterial and red algal phycobiliproteins. The structures of the remaining bilins were novel. The bilin at alpha-19, designated Cys-bilin 618, differed from PEB in having additional double bonds between C-2 and C-3 of ring A and between C-12' and C-12", i.e. an acryloyl substituent at C-12 of ring C. The doubly linked bilin at beta-50,61 designated DiCys-bilin 584, differed from doubly linked PEB (Schoenleber, R.W., Lundell, D.J., Glazer, A.N., and Rapoport, H. (1984) J. Biol. Chem. 259, 5481-5484) in possessing an acryloyl substituent at C-12 of ring C in place of a propionyl substituent. Similarly, the bilin at beta-158, designated Cys-bilin 584, differed from singly-linked PEB in possessing an acryloyl substituent at C-12 of ring C in place of a propionyl substituent. The three novel cryptomonad bilins join heme d1 and chlorophylls c1, c2, and c3 as the only known porphyrin-derived natural products with acryloyl substituents.     

R-phycocyanin II, a new phycocyanin occurring in marine Synechococcus species. Identification of the terminal energy acceptor bilin in phycocyanins

A new member of the phycocyanin family of phycobiliproteins, R-phycocyanin II (R-PC II) has been discovered in several strains of marine Synechococcus sp. R-PC II has absorption maxima at 533 and 554 nm, a subsidiary maximum at 615 nm, and a fluorescence emission maximum at 646 nm. It is the first phycoerythrobilin (PEB)-containing phycocyanin of cyanobacterial origin. The purified protein is made up of alpha and beta subunits in equal amounts and is in an (alpha beta)2 aggregation state. The alpha and beta subunits of this protein are homologous to the corresponding subunits of previously described C- and R-phycocyanins as assessed by amino-terminal sequence determination and analyses of sequences about sites of bilin attachment. R-PC II carries phycocyanobilin (PCB) at beta-84 and PEB at alpha-84 and beta-155 (residue numbering is that for C-phycocyanin), whereas in C-phycocyanin PCB is present at all three positions. In R-phycocyanin, the bilin distribution is alpha-84 (PCB), beta-84 (PCB), beta-155 (PEB). In both R-phycocyanin and R-phycocyanin II excitation at 550 nm, absorbed primarily by PEB groups, leads to emission at 625 nm from PCB. These comparative data support the conclusion that the invariant beta-84 PCB serves as the terminal energy acceptor in phycocyanins.     

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.     

Identification of the cAMP-dependent protein kinase and protein kinase C phosphorylation sites within the major intracellular domains of the beta 1, gamma 2S, and gamma 2L subunits of the gamma-aminobutyric acid type A receptor.

Gamma-aminobutyric acid Type A (GABAA) receptors are the major sites of synaptic inhibition in the central nervous system. These receptors are thought to be pentameric complexes of homologous transmembrane glycoproteins. Molecular cloning has revealed a multiplicity of different GABAA receptor subunits divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. Within the proposed major intracellular domain of these subunits, there are numerous potential consensus sites for protein phosphorylation by a variety of protein kinases. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC). The purified fusion protein of the intracellular domain of the beta 1 subunit was an excellent substrate for both PKA and PKC. PKA and PKC phosphorylated the beta 1 subunit fusion protein on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 409 in the intracellular domain of the beta 1 subunit to an alanine residue eliminated the phosphorylation of the beta 1 subunit fusion protein by both protein kinases. The purified fusion proteins of the major intracellular domain of the gamma 2S and gamma 2L subunits of the GABAA receptor were rapidly and stoichiometrically phosphorylated by PKC but not by PKA. The phosphorylation of the gamma 2S subunit occurred on serine residues on a single tryptic phosphopeptide. Site-directed mutagenesis of serine 327 of the gamma 2S subunit fusion protein to an alanine residue eliminated the phosphorylation of the gamma 2S fusion protein by PKC. The gamma 2L subunit is an alternatively spliced form of the gamma 2S subunit that differs by the insertion of 8 amino acids (LLRMFSFK) within the major intracellular domain of the gamma 2S subunit. The PKC phosphorylation of the gamma 2L subunit occurred on serine residues on two tryptic phosphopeptides. Site-specific mutagenesis of serine 343 within the 8-amino acid insert to an alanine residue eliminated the PKC phosphorylation of the novel site in the gamma 2L subunit. No phosphorylation of a purified fusion protein of the major intracellular loop of the alpha 1 subunit was observed with either PKA or PKC. These results identify the specific amino acid residues within GABAA receptor subunits that are phosphorylated by PKA and PKC and suggest that protein phosphorylation of these sites may be important in regulating GABAA receptor function.(ABSTRACT TRUNCATED AT 400 WORDS)     

Sulfhydryl groups of the F1 adenosine triphosphatase of Escherichia coli and the stoichiometry of the subunits

The distribution and total number of sulfhydryl groups present in the F1 adenosine triphosphatase of Escherichia coli were used to calculate the stoichiometry of the alpha-delta subunits. Titration with 5,5'-dithiobis (2-nitrobenzoate) gave 19.1 +/- 2.2 sulfhydryl groups/mol ATPase. Labeling with [14C]iodoacetamide and [14C]N-ethylmaleimide showed that 11.9, 3.1, 1.9, and 1.8 sulfhydryl groups per molecule of ATPase were associated with the alpha, beta, gamma, and delta subunits, respectively. The epsilon subunit was not labeled. Application of the method of Creighton [Nature (London) (1980) 284, 487-489] showed that 4, 1, and 2 sulfhydryl groups were present in the alpha, beta, and gamma subunits, respectively. This, together with published data for the delta subunit, allowed a subunit stoichiometry of alpha 3 beta 3 gamma delta to be calculated. The presence of four cysteinyl residues in the alpha subunit, as shown by several different methods, does not agree with the results of DNA sequencing of the ATPase genes [H. Kanazawa, T. Kayano, K. Mabuchi, and M. Futai (1981) Biochem. Biophys. Res. Commun. 103, 604-612; N. J. Gay and J. E. Walker (1981) Nucl. Acids Res. 9, 2187-2194] where three cysteinyl residues/alpha subunit have been found. It is suggested that post-translational modification of the alpha subunit to add a fourth cysteinyl residue might occur.     

Localization of the N-ethylmaleimide reactive cysteine in the beef heart mitochondrial ADP/ATP carrier protein

Alkylation of the ADP/ATP carrier protein in beef heart mitochondria by N-ethylmaleimide (NEM) results in inactivation of transport. One out of the four cysteinyl residues contained in 1 mol of carrier subunit of Mr 32 000 is alkylated by NEM. The identification of the alkylated residue to Cys-56 has been achieved by chemical and enzymatic cleavages. The chemical cleavages included cleavages at the nonalkylated cysteinyl residues by cyanide at alkaline pH and at methionyl residues by cyanogen bromide. Enzymatic cleavage involved the use of trypsin and chymotrypsin; the resulting peptides were resolved by high-performance liquid chromatography. Analysis of a small size [14C]NEM-labeled peptide obtained by tryptic and chymotryptic digestion of the [14C]NEM-labeled carrier protein yielded the following amino acid sequence: (Formula: see text) where X is probably a substituted lysine.