Biochemistry and Molecular Biology of Plant Hormones

P.J.J. Hooykaas, M.A. Hall, K.R. Libbenga

• Publisher: Elsevier Science
• Year: 1999
• Print ISBN: 9780444898258
• eBook ISBN: 9780080860947
• Topic: Biological Sciences
• Subtopic: Biochemistry
• Index: Biological Sciences

This book provides up-to-date coverage at an advanced level of a range of topics in the biochemistry and molecular biology of plant hormones, with particular emphasis on biosynthesis, metabolism and mechanisms of action. Each contribution is written by acknowledged experts in the field, providing definitive coverage of the field.No other modern book covers this subject matter at such an advanced level so comprehensively. It will be invaluable to university libraries and scientists in the plant biotechnology industries.

III – Hormone Perception and Transduction

Chapter 14

Molecular characteristics and cellular roles of guanine nucleotide binding proteins in plant cells

P.A. Millner and T.H. Carr, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

List of Abbreviations

ABP auxin binding protein

BSA bovine serum albumin

CTx cholera toxin

GNP guanine nucleoside phosphate

GTP7S guanosine 5′-0-(3-thiotriphosphate)

GppNHp β:γ-imidoguanosine diphosphate

GNRP guanine nucleotide regulatory protein

GAP GTPase activating protein

PLC phospholipase C

IP₃ inositol 1,4,5-trisphosphate

PI phosphatidyl inositol

PTx pertussis toxin

AC adenylate cyclase

7TMS seven transmembrane span

1. Signal transducing GTPases within animal and fungal cells

1.1. Major subclasses

Monomeric/small G-proteins: The archetypal small G-protein is that encoded by the ras gene which, in animal cells at least, is a highly conserved 21 kDa monomeric protein, ras^p21 (p21^ras). The ras proteins were discovered initially because of their transforming ability in the Harvey and Kirsten strains of rat sarcoma virus. All members of the ras family bind and hydrolyse GTP, becoming “active” in the presence of the triphosphate and “inactive” upon hydrolysis of it. Interestingly, ras proteins possess extremely low intrinsic hydrolysis rates (<0.01 min^–1) as compared to their distant cousins, the heterotrimeric G-proteins (3–5 min^–1). Thus the ras proteins are dependent on several types of “partner” proteins: GAPs (GTPase activating proteins), GNRPs (guanine nucleotide release proteins) and GDIs (guanine nucleotide dissociation inhibitors) to regulate GTP hydrolysis, nucleotide exchange and release [20,93]. A vital prerequisite for the signalling role of the ras proteins is lipid modification, with an acyl or prenyl group being attached at a C-terminal cysteine residue, allowing anchorage to the inner face of the plasma membrane [29,59].

Ras is known to be involved in the regulation of cellular proliferation and terminal differentiation [7,40]. In mammals, ras is activated by growth-factor-receptor tyrosine kinases and other tyrosine kinases. Some of these kinases phosphorylate the Shc protein and phosphorylated Shc plus autophosphorylated receptor proteins bind the SH2 domain of Grb2. The resulting complex recruits Sos, a guanine nucleotide dissociation stimulator, to the plasma membrane and Sos promotes release of GDP from inactive Ras, allowing GTP to bind. The now active Ras can directly stimulate effector proteins further down the transduction cascade (see reference [93]) and references therein). Some of the downstream proteins are believed to include the mitogen-activated protein kinases (MAPKs) and other serine/threonine protein kinases such as Raf [21,93]. In S. cerevisiae the two RAS proteins, which at approximately 40 kDa are somewhat larger than their mammalian counterparts [121], regulate cAMP levels by stimulating an adenylate cyclase, possibly directly, [147] though this is not the case in all other eukaryotes studied thus far.

Many other proteins closely related to ras are now known and are considered as members of the “ras superfamily” which numbers > 100 members. Within this superfamily are several subgroups, with Ras being joined by the Ran/Rac, Ypt/Rab and Rho proteins. The cellular functions of these proteins are exceedingly diverse including such processes as vesicular trafficking, cytoskeletal control and NADPH oxidase function. Clearly, such important proteins have plant homologues which will be discussed later.

Heterotrimeric G-proteins: A number of excellent reviews are already extant concerning the heterotrimeric G-protein family covering topic areas on their structure, molecular connections and functions [16,23,24,33,34,37,44,49,53,73,87,107,125,140]. In the present review we shall only attempt to give an overview.

The heterotrimeric G-proteins are comprised of a trio of distinct subunits, termed α, β and γ respectively. The α subunit, which is usually within the Mr range 35–45 kDa or so, is responsible for coupling to the respective receptor (see Section 1.4) after the latter has bound its cognate ligand. The same subunit is also responsible for subsequent interaction with its effector or effector systems. Structurally, the α subunits, which now number more than 80 determined sequences, display a high degree of conservation within certain domains, e.g. the γ-phosphate binding domain. This attribute has been utilised in a number of cases to develop antibodies, directed against synthetic peptides whose sequences correspond to these domains, as probes for specific G-protein subtypes [60,104]. Such antisera have also found use in identifying candidate plant G-proteins (Section 2.3). Other regions within the primary amino acid sequence are less conserved. Via a panoply of techniques these regions have been identified as specifying interaction of G_α with its receptor or effector(s). Amongst these efforts have been the construction of chimaeric G_α subunits which incorporate portions of the Gs_α and Gi_α sequences [11,94,160] and of coupling defective mutants [145]. Other work involved ADP-ribosylation by cholera [72,150] and pertussis toxins [149,155]. In the latter cases, for example, the ADP-ribose moiety was known to be attached to a near C-terminal cysteine and was shown to decrease the interaction of various Gi_α subtypes with the cognate receptor. Finally, a number of studies have shown that synthetic peptides corresponding in sequences to regions of the Gt_α and Gs_α respectively [31,61,114,115] are effective at modulating either receptor/G_α coupling or G_α/effector coupling. This body of work, taken together has led to the indication that the extreme C-terminal portion contributes substantially to receptor/G_α coupling whilst interaction of the G-protein with its effector is defined by regions of sequence which are more internal, although still within the C-terminal half of the protein [37]. Finally, in the past few years, many of the predictions made on the basis of biochemical and molecular genetic approaches have been borne out by the determination, to high resolution of the crystal structures of the G_α-subunit [84,137] and the Ga_αβγ heterotrimer [85].

With respect to the other G-protein subunits, the relatively small number of G_β found so far have been found to be extremely highly conserved [52,54,144] whilst G_γ subunits are somewhat diverse in structure [55,68,127]. In vivo, and in vitro the G_β and G_γ subunits are always found tightly associated as a complex, whose existence may be important to the stabili...

Table of contents

• Cover image
• Title page
• Table of Contents
• New Comprehensive Biochemistry
• Copyright page
• Preface
• List of contributors*
• Other volumes in the series
• I – Introduction and Methodology
• II – Control of Hormone Synthesis and Metabolism
• III – Hormone Perception and Transduction