NORMAL GENOMIC

• Hisbergues M, Gaitatzes CG, Joset F, Bedu S, Smith TF
• A noncanonical WD-repeat protein from the cyanobacterium Synechocystis PCC6803: structural and functional study.
• Protein Sci. 2001; 10: 293-300
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• SYNECHOCYSTIS: PCC6803 possesses several open reading frames encoding putative WD-repeat proteins. One, the Hat protein, is involved in the control of a high-affinity transport system for inorganic carbon that is active when the cells are grown under a limiting concentration of this carbon substrate. The protein is composed of two major domains separated by a hydrophobic linker region of 20 amino acid residues. The N-terminal domain of Hat has no homolog in standard databases and does not display any particular structural features. Eleven WD repeats have been identified in the C-terminal moiety. The region encompassing the four terminal WD repeats is essential for growth under a limiting inorganic carbon regime. The region encompassing the two most terminal WD repeats is required for the activity of the high-affinity transport system. However, because the Hat protein is located in the thylakoids, it should not be itself an element of the transport system. The structural organization of the WD-containing domain of Hat was modeled from the crystal structure of the G protein beta subunit (with seven WD repeats) and of hemopexin (a structural analog with four blades). Functional and structural data argue in favor of an organization of the Hat WD moiety in two subdomains of seven and four WD repeats. The C-terminal 4-mer subdomain might interact with another, yet unknown, protein/peptide. This interaction could be essential in modulating the stability of the 4-mer structure and, thus, the accessibility of this subdomain, or at least of the region encompassing the last two WD repeats.

• Kasahara S, Wang P, Nuss DL
• Identification of bdm-1, a gene involved in G protein beta-subunit function and alpha-subunit accumulation.
• Proc Natl Acad Sci U S A. 2000; 97: 412-7
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• Targeted disruption of Galpha and Gbeta genes has established the requirement of an intact G protein signaling pathway for optimal execution of several important physiological processes, including pathogenesis, in the chestnut blight fungus Cryphonectria parasitica. We now report the identification of a G protein signal transduction component, beta disruption mimic factor-1, BDM-1. Disruption of the corresponding gene, bdm-1, resulted in a phenotype indistinguishable from that previously observed after disruption of the Gbeta subunit gene, cpgb-1. The BDM-1 deduced amino acid sequence contained several significant clusters of identity with mammalian phosducin, including a domain corresponding to a highly conserved 11-amino acid stretch that has been implicated in binding to the Gbetagamma dimer and two regions of defined Gbeta/phosducin contact points. Unlike the negative regulatory function proposed for mammalian phosducin, the genetic data presented in this report suggest that BDM-1 is required for or facilitates Gbeta function. Moreover, disruption of either bdm-1 or cpgb-1 resulted in a significant, posttranscriptional reduction in the accumulation of CPG-1, a key Galpha subunit required for a range of vital physiological processes.

• Stoytcheva Z, Joshi B, Spizek J, Tichy P
• WD-repeat protein encoding genes among prokaryotes of the Streptomyces genus.
• Folia Microbiol (Praha). 2000; 45: 407-13
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• Southern hybridization with probes designed for detection of WD-repeats coding sequences gave positive results in 21 streptomycete strains indicating that WD-repeats encoding genes are massively spread among streptomycetes. One of them, the wdlA gene of Streptomyces lincolnensis, codes for a 971 amino acid protein with seven WD-repeats in its C-terminus, two transmembrane domains and an ATP/GTP binding site upstream of the WD-repeat region.

• Kasahara S, Nuss DL
• Targeted disruption of a fungal G-protein beta subunit gene results in increased vegetative growth but reduced virulence.
• Mol Plant Microbe Interact. 1997; 10: 984-93
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• Targeted disruption of two G-protein alpha subunit genes in the chestnut blight fungus Cryphonectria parasitica revealed roles for the Gi alpha subunit CPG-1 in fungal reproduction, virulence, and vegetative growth. A second G alpha subunit, CPG-2, was found to be dispensable for these functions. We now report the cloning and targeted disruption of a C. parasitica G-protein beta subunit gene. The deduced amino acid sequence encoded by this gene, designated cpgb-1, was found to share 66.2, 65.9, and 66.7% amino acid identity with G beta homologues from human, Drosophila, and Dictyostelium origins, respectively, but only 39.7% identity with the Saccharomyces cerevisiae G beta homologue STE4 product. Low stringency Southern hybridization failed to detect any related G beta subunit genes in C. parasitica. Targeted disruption of cpgb-1 resulted in several of the changes previously reported to accompany disruption of the C. parasitica Gi alpha subunit gene cpg-1. These included very significant reductions in pigmentation, asexual sporulation, and virulence. In contrast to results obtained for Gi alpha gene disruption, the reduction in virulence resulting from the disruption of a G beta gene was accompanied by increased, rather than decreased, vegetative growth on synthetic medium. The relevance of these results to mechanisms of fungal virulence is considered.

• Garcia-Higuera I, Thomas TC, Yi F, Neer EJ
• Intersubunit surfaces in G protein alpha beta gamma heterotrimers. Analysis by cross-linking and mutagenesis of beta gamma.
• J Biol Chem. 1996; 271: 528-35
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• Heterotrimeric guanine nucleotide binding proteins (G proteins) are made up of alpha, beta, and gamma subunits, the last two forming a very tight complex. Stimulation of cell surface receptors promotes dissociation of alpha from the beta gamma dimer, which, in turn, allows both components to interact with intracellular enzymes or ion channels and modulate their activity. At present, little is known about the conformation of the beta gamma dimer or about the areas of beta gamma that interact with alpha. Direct information on the orientation of protein surfaces can be obtained from the analysis of chemically cross-linked products. Previous work in this laboratory showed that 1,6-bismaleimidohexane, which reacts with cysteine residues, specifically cross-links alpha to beta and beta to gamma (Yi, F., Denker, B. M., and Neer, E. J. (1991) J. Biol. Chem. 266, 3900-3906). To identify the residues in beta and gamma involved in cross-linking to each other or to alpha, we have mutated the cysteines in beta 1, gamma 2, and gamma 3 and analyzed the mutated proteins by in vitro translation in a rabbit reticulocyte lysate. All the mutants were able to form beta gamma dimers that could interact with the alpha subunit. We found that 1,6-bismaleimidohexane can cross-link beta 1 to gamma 3 but not to gamma 2. The cross-link goes from Cys25 in beta 1 to Cys30 in gamma 3. This cysteine is absent from any of the other known gamma isoforms and therefore confers a distinctive property to gamma 3. The beta subunit in the beta 1 gamma 2 dimer can be cross-linked to an unidentified protein in the rabbit reticulocyte lysate, generating a product slightly larger than cross-linked beta 1 gamma 3. The beta subunit can also be cross-linked to alpha, giving rise to two products on SDS-polyacrylamide gel electrophoresis, both of which were previously shown to be formed by cross-linking beta to Cys215 in alpha o (Thomas, T. C., Schmidt, C. J., and Neer, E. J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10295-10299). Mutation of Cys204 in beta 1 abolished one of these two products, whereas mutation of Cys271 abolished the other. Because both alpha-beta cross-linked products are formed in approximately equal amounts, Cys204 and Cys271 in beta are equally accessible from Cys215 in alpha o. Our findings begin to define intersubunit surfaces, and they pose structural constraints upon any model of the beta gamma dimer.

• Lee RH, Lieberman BS, Yamane HK, Bok D, Fung BK
• A third form of the G protein beta subunit. 1. Immunochemical identification and localization to cone photoreceptors.
• J Biol Chem. 1992; 267: 24776-81
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• Vertebrate retinal cones play a major role in both photopic vision and color perception. Although the molecular mechanism of visual excitation in the cone is not as well understood as in the rod, it is generally thought to involve a cone-specific G protein (cone transducin) that couples the cone visual pigment to a cGMP phosphodiesterase. Like all other G proteins, cone transducin is most likely a heterotrimer consisting of G alpha, G beta, and G gamma subunits. A G alpha subunit of cone transducin has been localized to the outer segment of bovine cones, but its associated G beta and G gamma subunits are unknown. To identify the G beta subunit involved in the phototransduction process of cones, we have developed a panel of antipeptide antisera against the most diverse region of the amino acid sequences encoded by G beta 1, G beta 2, and G beta 3 cDNAs and used them to determine the distribution of the G beta isoforms in different retinal preparations. We found that the G beta 3 subunit is present in bovine retinal transducin and phosducin-T beta gamma complex preparations which were previously thought to contain only G beta 1. Analysis of its subcellular distribution indicated that G beta 3 is predominantly cytoplasmic. Immunocytochemical staining of bovine retinal sections with the anti-G beta 3 antiserum further revealed a specific localization of G beta 3 in cones but not in rods. In contrast, anti-G beta 1 antiserum stained only the rods. These results suggest that G beta 3 is the G beta subunit of cone transducin and confirms the proposition that rods and cones utilize distinct signaling proteins for phototransduction.