GTPases are conserved molecular switches, built according to a common structural design. Rapidly accruing knowledge of individual GTPases--crystal structures, biochemical properties, or results of molecular genetic experiments--support and generate hypotheses relating structure to function in other members of the diverse family of GTPases.

The GTPase superfamily: conserved structure and molecular mechanism

Proteins that bind and hydrolyse GTP are being discovered at a rapidly increasing rate. Each of these many GTPases acts as a molecular switch whose 'on' and 'off' states are triggered by binding and hydrolysis of GTP. Conserved structure and mechanism in myriad versions of the switch--in bacteria, yeast, flies and vertebrates--suggest that all derive from a single primordial protein, repeatedly modified in the course of evolution to perform a dazzling variety of functions.

The GTPase superfamily: a conserved switch for diverse cell functions

THE definition of cellular organelles has evolved over the last hundred years largely driven by morphologic observations, but more recently has been supplemented and complemented by functional and biochemical studies (Palade, 1975) . Thus, organelles are now identified both by their morphology and by the set ofcomponents that comprise them . Determining how organelle identity is established and maintained and how newly synthesized protein and membrane are sorted to different organelles are the central issues of organellogenesis . Essential to the many cellular functions that take place within the central vacuolar system (which consists ofthe ER, Golgi apparatus, secretory vesicles, endosomes, and lysosomes) is membrane traffic which mediates the exchange of components between different organelles . There are two critical characteristics of membrane traffic . First, only certain sets oforganelles exchange membrane and the patterns of this exchange define what are called membrane pathways . Second, multiple pathways intersect at specific points within the central vacuolar system . For specific components to "choose" the correct pathway at such points of crossing, mechanisms exist to impose choices on specific molecules . This process is called sorting . The characteristicsofeachorganelle within the central vacuolar system are likely to be intimately tied to the properties ofmembrane traffic . An imbalance in the magnitude ofmembrane input into and egress from an organelle would have profound effects on the size ofthat compartment . In addition, failures in sorting or aberrations in targeting pathways would be expected to profoundly affect the identity of individual organelles . Recently, the relationship between the control of membrane traffic and the maintenance of organelle structure has been investigated with the use ofa remarkable drug, brefeldin A (BFA).' In this review we will summarize recent findings with BFA and propose some speculative models concerning the mechanism and regulation ofmembrane traffic within the central vacuolar system .

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Brefeldin A: insights into the control of membrane traffic and organelle structure.

Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain

The structure of a heterotrimeric G protein reveals the mechanism of the nucleotide-dependent engagement of the alpha and beta gamma subunits that regulates their interaction with receptor and effector molecules. The interaction involves two distinct interfaces and dramatically alters the conformation of the alpha but not of the beta gamma subunits. The location of the known sites for post-translational modification and receptor coupling suggest a plausible orientation with respect to the membrane surface and an activated heptahelical receptor.

The 2.0 Å crystal structure of a heterotrimeric G protein

The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding.

The regulatory component of adenylate cyclase. Purification and properties.

The regulatory component (G/F) of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from rabbit liver plasma membranes has been purified essentially to homogeneity. The purification was accomplished by three chromatographic procedures in sodium cholate-containing solutions, followed by three steps in Lubrol-containing solutions. The specific activity of G/F was enriched 2000-fold from extracts of membranes to 3-4 mumol x min-1 x mg-1 (reconstituted adenylate cyclase activity). Purified G/F reconstitutes guanine nucleotide-, fluoride-, and hormone-stimulated adenylate cyclase activity in the adenylate cyclase-deficient variant of S49 murine lymphoma cells. G/F also recouples hormonal stimulation of the enzyme in the uncoupled variant of S49. Preparations of pure G/F contain three polypeptides with approximate molecular weights of 52,000, 45,000, and 35,000. The active G/F protein behaves as a multisubunit complex of these polypeptides. Treatment of G/F with [32P]NAD+ and cholera toxin covalently labels the molecular weight 52,000 and 45,000 polypeptides with 32P.

https://www.pnas.org/content/pnas/77/11/6516.full.pdf

Purification of the regulatory component of adenylate cyclase

The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution of the activated 45,000-dalton (alpha) subunit.

The regulatory component of adenylate cyclase (G/ F) contains the site(s) for activation of the enzyme by guanine nucleotides. We have developed methods for the assessment of binding of radiolabeled guanosine-5’(3-0-thio)triphosphate (GTPyS) and guanosine-5‘-(P,yimin0)triphosphate (Gpp(NH)p) to homogeneous preparations of G/F. These methods detect binding specifically to the activating site of G/F as judged by the following criteria. 1) The kinetics of activation and binding are identical. 2) Apparent Kd values for [%] GTPyS (0.7 PM) and I3HJGpp(NH)p (5 PM) agree with K, values for activation of G/F. 3) Apparent Kd values for nonactivating nucleotides, determined by competition for binding of GTPyS, agree with Ki values for inhibition of activation by GTPyS. 4) Both binding and activation depend on divalent metal ions with the same specificity. Binding of [36S]GTPyS or [3H]Gpp(NH)p reveals a single class of sites with an apparent stoichiometry of 1 mol of sites/mol of G/F (Mr = 80,000). Photoaffinity labeling of G/F with [y-32P]8-azido guanosine-5’-triphosphate specifically incorporates 32P into the 52,000- and 45,000-dalton subunits of G/F. Activation of G/F by GTPyS or Gpp(NH)p does not conform to simple Michaelis-Menten kinetics. While Hofstee and Scatchard analyses of activation and binding isotherms at apparent steady state appear to behave in a Michaelis-Menten manner, there is no reversal of activation or binding under activating conditions and the rate constant for activation is unchanged from 10 mi to 100 PM GTPyS. Reversal of binding and activation, which occurs only in the absence of divalent cation, is not a first order process. These anomalous kinetics are explained by a model in which rapid equilibrium binding of GTPyS precedes a dissociation of the 45,000- and 35,000-dalton subunits of G/F. This latter reaction is the rate-limiting step in the activation pathway. This model predicts an inhibitory function of the 35,000-dalton subunit, which we have confirmed with the resolved protein.

M D Smigel

Papers

The guanine nucleotide activating site of the regulatory component of adenylate cyclase. Identification by ligand binding.

The regulatory component of adenylate cyclase. Purification and properties.

Purification of the regulatory component of adenylate cyclase

The subunits of the stimulatory regulatory component of adenylate cyclase. Resolution of the activated 45,000-dalton (alpha) subunit.

The Guanine Nucleotide Activating Site of the Regulatory Component of Adenylate Cyclase