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GTP-Binding Proteins in Plants: New Members of an Old Family

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GTP-Binding Proteins in Plants: New Members of an Old Family Plant Molecular Biology 26: 1611-1636, 1994. © 1994 Kluwer Academic Publishers. Printed in Belgium. 1611 GTP-binding proteins in plants: new members of an old family Hong Ma Cold Spring Harbor Laboratory, CoM Spring Harbor, NY 11724, USA Received and accepted 13 June 1994 Key words: guanine nucleotide-binding proteins, heterotrimeric G proteins, small G proteins, biochemical detection, cDNAs, expression, yeast complementation, transgenic plants Abstract Regulatory guanine nucleotide-binding proteins (G proteins) have been studied extensively in animal and microbial organisms, and they are divided into the heterotrimeric and the small (monomeric) classes. Heterotrimeric G proteins are known to mediate signal responses in a variety of pathways in animals and simple eukaryotes, whiole small G proteins perform diverse functions including signal transduction, secretion, and regulation of cytoskeleton. In recent years, biochemical analyses have produced a large amount of information on the presence and possible functions of G proteins in plants. Further, molecular cloning has clearly demonstrated that plants have both heterotrimeric and small G proteins. Although the functions of the plant heterotrimeric G proteins are yet to be determined, expression analysis of an Arabidopsis G~ protein suggests that it may be involved in the regulation of cell division and differen- tiation. In contrast to the very few genes cloned thus far that encode heterotrimeric G proteins in plants, a large number of small G proteins have been identified by molecular cloning from various plants. In addition, several plant small G proteins have been shown to be functional homologues of their coun- terparts in animals and yeasts. Future studies using a number of approaches are likely to yield insights into the role plant G proteins play. Introduction protein (Gs) that mediates the hormonal stimu- lation of adenylate cyclase, and is widespread in GTP-binding regulatory proteins (for simplicity, animal cells [59]. In this case, the binding of the referred to as G proteins hereafter) are members signal epinephrine to its receptor triggers the ac- of a large family of guanine nucleotide binding tivation of Gs, which then activates adenylate cy- proteins found in all eukaryotes. On the basis of clase. The synthesis of cAMP in turn leads to a subunit composition and size, G proteins have cascade of protein phosphorylations and dephos- been classified as heterotrimeric or small (mono- phorylations, which regulate the activity of a meric) G proteins. Heterotrimeric G proteins, variety of proteins. Another well characterized consisting of ~, fl, and ~ subunits, generally relay example of heterotrimeric G protein is the trans- information from membrane receptors to intra- ducins (Gt) , which transmit visual signals in ver- cellular effectors [ 16, 17, 59, 144, 155]. One of the tebrates [59]. The Gt-coupled receptor is the best studied heterotrimeric G proteins is the G light-activated rhodopsin, and the Gt-activated [375] 1612 effector is cGMP phosphodiesterase. Therefore, siae have demonstrated an important role for Ras G t mediates the light-activated hydrolysis of in the regulation of cellular growth; in particular, cGMP, which in turn regulates Na +/Ca 2 + chan- Ras regulate the activity of adenylate cyclase in nels in the photoreceptors, altering membrane po- yeast, and much (but not all) of the Ras function tentials [160]. In animals, extensive biochemical is mediated by cAMP in yeast [177, 178]. A and pharmacological studies have accumulated a variety of studies in recent years have identified a large amount of information on many additional large number of different, but related, small G heterotrimeric G proteins and their functions [59, proteins functioning in a variety of processes, 144]. In recent years, molecular cloning has iden- from signal transduction to controlling protein tified genes for previously known G proteins, as secretion, from regulating cytoskeletal functions well as genes for new types of heterotrimeric G to organizing membranes [63, 64]. proteins in both mammals and other animals [87, Since the late 1980s, biochemical, physiologi- 155]. Furthermore, a combination of genetic, bio- cal and molecular approaches have been success- chemical and molecular analyses have uncovered fully used to demonstrate the presence of G pro- G protein functions in simple eukaryotes [ 16, 17, teins in plants. For comparison, this article will 87, 155]. The sizes of the ~ subunits range from summarize briefly G proteins in animals and 35 to 45 kDa, those of the fi and 7 subunits are simple eukaryotes, and focus the remainder of the generally of 35-36 and 8-10 kDa, respectively. In discussion on the recent results on G proteins in addition, recent biochemical studies have uncov- plants. Other GTP-binding proteins, including ered novel proteins that are much larger (66-74 tubulins and translation factors, will not be dis- kDa) than known Gc~'s, yet have some charac- cussed here, although they perform important cel- teristics of Ga subunits, such as GTP-binding, lular functions. Other recent reviews on plant G cross-reactivity with antisera against a conserved proteins offer different emphases and perspectives Gc~ peptide, and interaction with G-protein- [86, 94, 163]. coupled membrane receptors [73, 79, 80, 125, 156]. It is not known, however, how structurally similar these new GTP-binding proteins are to G protein structures and functions in animals and known Ge subunits, and whether they interact simple eukaryotes with either known or novel Gfl 7 subunits. The small G proteins include a large number of Heterotrimeric G proteins molecules, from 20 to 30 kDa in size, and have very diverse functions [63, 64]. The discovery Heterotrimeric G proteins were first identified in that the proto-oncogene ras encodes a small mammalian signal transduction pathways [87, GTP-binding protein opened a new chapter in the 155]. In addition to the aforementioned Gs, there history of G proteins. Although the precise bio- are three inhibitory G proteins (Gi(1-3)) which chemical function of mammalian ras is still not mediate the hormonal inhibition of adenylate cy- clear, increasing evidence suggests that it medi- clase activity. Furthermore, there are two trans- ates signals received by membrane-receptor ty- ducins, Gtl and Gt2 , which transmit visual signals rosine kinase(s) and transmits them through other to membrane potentials in rod and cone photo- proteins to protein kinases, in particular the MAP receptor cells, respectively. The genes encoding kinase cascade [64, 109, 149, 164]. Genetic and the e subunits of these G proteins have been iso- molecular studies in invertebrate animals have lated [87]; the Gi's and Gt's are more similar to also provided strong support for this signalling each other at the amino acid sequence level than pathway, which is important for the regulation of they are to Gs (Fig. 1). Another Gc~, Go, was specific cellular differentiations [ 145, 157]. Mo- discovered which is most similar to Gi in lecular, genetic and biochemical analyses of the sequence. Other mammalian Ge genes have been Ras proteins in the yeast Saecharomyees cerevi- isolated that are structurally and functionally re- [376] 1613 Gi Gq G12 Gs Yeast Plant I I I ]1 I I I I 1[-----7 Identity (%) ¢q 100 -- aOOOota OOO O 80 -- 2 60 -- 40 -- 20 Fig. 1. Comparison of heterotrimeric G protein ~ subunits.The similarity tree is based on the percentage of amino acid sequence identity, and is modified from Fig. 2 of Simon et al. [ 155]; the additional amino acids in Gs, GoLf, and the yeast ScG1 were not included in the comparison, as described in Lochrie and Simon [103]. The DG's are from Drosophila melanogaster, DictyG2 is from Dictyostelium discoideum, ScG 1 from Saccharomyces cerevisiae, SpG 1 from Schizosaccharomycespombe, AtG 1 from A rabidopsis thaliana, ToG1 from tomato, and the other from mammals. lated to Gs (Golf, for olfactory response) or G t can serve as a site for ADP ribosylation by chol- (gustducin, for taste sensation) [ 111, 155]. More- era toxin; however, only some GT's are known to over, two new classes of G~ have been isolated be modified by cholera toxin while others are not recently: the Gq and the G12/G13 classes [2, 158, [ 155]. ADP ribosylation by cholera toxin blocks 159, 179]. At least some of these new G proteins GTPase activity, resulting in an activated form of are involved in the regulation of phospholipase C the c~ subunit (Fig. 2A). Some ~ subunits, includ- [155]. Figure 1 shows the amino acid similarity ing Gi's and Gt's, have a site (a cysteine residue between different Gc~ proteins. Genes encoding near the C-terminus) for a similar modification by homologues of all major types of mammalian G~ pertussis toxin. The ADP ribosylation by pertus- proteins (Fig. 1) have also been isolated from sis toxin interferes with the interaction between Drosophila [36, 129, 132, 135-137, 158], and G~ and the receptor, and blocks the exchange of genes for the Gs and Go types of G~ have been GDP for GTP, rendering the protein unable to be isolated from Caenorhabditis elegans [51, 102]. activated by the receptor (Fig. 2A, see below for Among simple eukaryotes, the yeasts S. cerevisiae the mechanism of G protein action). and Schizosaccharomyces pombe each have two Molecular cloning has identified several mam- G~ genes [40, 76, 115, 118, 119, 124]. Dictyoste- malian genes encoding at least 4 different fl sub- lium has as many as eight different ~ subunits, units and 5 7 subunits [74, 155]. Genes for fl G~I-G~8 [52, 62, 92, 133, 182]. subunits have also been isolated from inverte- Heterotrimeric G protein ~ subunits have con- brate animals squid [ 146], Drosophila [ 184, 185] served consensus regions for GTP binding and and C. elegans [165]. In addition, the budding GTPase activity [ 17, 87, 155]. A conserved argi- yeast S. cerevisiae STE4 and STE18 genes were nine residue is found in all known G~'s, and it found to encode fl and 7 subunits, respectively [377] 1614 Llgaud 84].
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