Role of coatomer and phospholipids in GTPase-activating protein-dependent hydrolysis of GTP by ADP-ribosylation factor-1

The binding of the coat protein complex, coatomer, to the Golgi is mediated by the small GTPase ADP-ribosylation factor-1 (ARF1), whereas the dissociation of coatomer, requires GTP hydrolysis on ARF1, which depends on a GTPase-activating protein (GAP). Recent studies demonstrate that when GAP activity is assayed in a membrane-free environment by employing an amino-terminal truncation mutant of ARF1 (Delta17-ARF1) and a catalytic fragment of the ARF GTPase-activating protein GAP1, GTP hydrolysis is strongly stimulated by coatomer (Goldberg, J., (1999) Cell 96, 893-902). In this study, we investigated the role of coatomer in GTP hydrolysis on ARF1 both in solution and in a phospholipid environment. When GTP hydrolysis was assayed in solution using Delta17-ARF1, coatomer stimulated hydrolysis in the presence of the full-length GAP1 as well as with a Saccharomyces cerevisiae ARF GAP (Gcs1) but had no effect on hydrolysis in the presence of the phosphoinositide dependent GAP, ASAP1. Using wild-type myristoylated ARF1 loaded with GTP in the presence of phospholipid vesicles, GAP1 by itself stimulated GTP hydrolysis efficiently, and coatomer had no additional effect. Disruption of the phospholipid vesicles with detergent resulted in reduced GAP1 activity that was stimulated by coatomer, a pattern that resembled Delta17-ARF1 activity. Our findings suggest that in the biological membrane, the proximity between ARF1 and its GAP, which results from mutual binding to membrane phospholipids, may be sufficient for stimulation of ARF1 GTPase activity.     

Active ADP-ribosylation factor-1 (ARF1) is required for mitotic Golgi fragmentation

In mammalian cells the Golgi apparatus undergoes an extensive disassembly process at the onset of mitosis that is believed to facilitate equal partitioning of this organelle into the two daughter cells. However, the underlying mechanisms for this fragmentation process are so far unclear. Here we have investigated the role of the ADP-ribosylation factor-1 (ARF1) in this process to determine whether Golgi fragmentation in mitosis is mediated by vesicle budding. ARF1 is a small GTPase that is required for COPI vesicle formation from the Golgi membranes. Treatment of Golgi membranes with mitotic cytosol or with purified coatomer together with wild type ARF1 or its constitutive active form, but not the inactive mutant, converted the Golgi membranes into COPI vesicles. ARF1-depleted mitotic cytosol failed to fragment Golgi membranes. ARF1 is associated with Golgi vesicles generated in vitro and with vesicles in mitotic cells. In addition, microinjection of constitutive active ARF1 did not affect mitotic Golgi fragmentation or cell progression through mitosis. Our results show that ARF1 is active during mitosis and that this activity is required for mitotic Golgi fragmentation.     

Mechanism of GTP hydrolysis at microtubule ends

The kinetics of tubulin subunits incorporation into microtubules and the kinetics of inorganic phosphate release have been measured in parallel. Correlation of the two measurements indicates that the tubulin GTPase activity is due to GTP hydrolysis and exchange at the end of the microtubules. In some cases where the free GTP available in the medium is in-sufficient the rate of GTP hydrolysis is limited by the rate of tubulin-GTP association at the end of the microtubules. The affinity constant of GTP for the microtubule end appears to be 100 times lower than the affinity constant of the tubulin-GTP complex.     

Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission

The mechanisms by which the coat complex II (COPII) coat mediates membrane deformation and vesicle fission are unknown. Sar1 is a structural component of the membrane-binding inner layer of COPII (Bi, X., R.A. Corpina, and J. Goldberg. 2002. Nature. 419:271-277). Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes. Although Sar1 activation leads to constriction of endoplasmic reticulum (ER) membranes, progression to effective vesicle fission requires a functional Sar1 NH2 terminus and guanosine triphosphate (GTP) hydrolysis. Inhibition of Sar1 GTP hydrolysis, which stabilizes Sar1 membrane binding, resulted in the formation of coated COPII vesicles that fail to detach from the ER. Thus Sar1-mediated GTP binding and hydrolysis regulates the NH2-terminal tail to perturb membrane packing, promote membrane deformation, and control vesicle fission.     

Significance of GTP hydrolysis in Ypt1p-regulated endoplasmic reticulum to Golgi transport revealed by the analysis of two novel Ypt1-GAPs

We here report on the identification and detailed biochemical characterization of two novel GTPase-activating proteins, Gyp5p and Gyp8p, whose efficient substrate is Ypt1p, a Ypt/Rab-GTPase essential for endoplasmic reticulum-to-Golgi trafficking in yeast. Gyp5p accelerated the intrinsic GTPase activity of Ypt1p 4.2 x 10(4)-fold and, surprisingly, the 40-fold reduced GTP hydrolysis rate of Ypt1(Q67L)p 1.5 x 10(4)-fold. At steady state, the two newly discovered GTPase-activating proteins (GAPs) as well as the previously described Gyp1p, which also uses Ypt1p as the preferred substrate, display different subcellular localization. To add to an understanding of the significance of Ypt1p-bound GTP hydrolysis in vivo, yeast strains expressing the GTPase-deficient Ypt1(Q67L)p and having different Ypt1-GAP genes deleted were created. Depending on the genetic background, different mutants exhibited growth defects at low temperature and, already at permissive temperature, various morphological alterations resembling autophagy. Transport of proteins was not significantly impaired. Growth defects of Ypt1(Q67L)-expressing cells could be suppressed on high expression of all three Ypt1-GAPs. We propose that permanently active Ypt1p leads to increased vesicle fusion, which might induce previously unnoticed autophagic degradation of exaggerated membrane-enclosed structures. The data indicate that hydrolysis of Ypt1p-bound GTP is a prerequisite for a balanced vesicle flow between endoplasmic reticulum and Golgi compartments.     

Binding of GDP to a ribosomal protein after elongation factor-2 dependent GTP hydrolysis.

Incubation of 80S ribosomes with a substoichiometric amount of [alpha-32P]GTP and with eEF-2 resulted in the specific labeling of one ribosomal protein which migrated very close to the position of the acidic phosphoprotein P2 from the 60S subunit in two-dimensional isofocusing-SDS gel electrophoresis. Localization of protein P2 in this electrophoretic system was ascertained by correlation with its position in the standard two-dimensional acidic-SDS gel electrophoresis after its specific phosphorylation by casein kinase II. Labeling of the ribosomal protein was dependent on the presence of eEF-2, and could be attributed to [alpha-32P]GDP binding from the results of chase experiments and HPLC identification, this binding being very likely responsible for the slight shift in the electrophoretical position of the protein. Incubation of ribosomes with tRNA(Phe) in the absence of mRNA induced the release of the bound GDP.     

Regulation of GTP hydrolysis prior to ribosomal AUG selection during eukaryotic translation initiation

Genetic studies in yeast have shown that the translation initiation factor eIF5 plays an important role in the selection of the AUG start codon. In order to ensure translation fidelity, the hydrolysis of GTP bound to the 40S preinitiation complex (40S.Met-tRNA(i).eIF2.GTP), promoted by eIF5, must occur only when the complex has selected the AUG start codon. However, the mechanism that prevents the eIF5-promoted GTP hydrolysis, prior to AUG selection by the ribosomal machinery, is not known. In this work, we show that the presence of initiation factors eIF1, eIF1A and eIF3 in the 40S preinitiation complex (40S.eIF1.eIF1A.eIF3.Met-tRNA(i).eIF2.GTP) and the subsequent binding of the preinitiation complex to eIF4F bound at the 5'-cap structure of mRNA are necessary for preventing eIF5-promoted hydrolysis of GTP in the 40S preinitiation complex. This block in GTP hydrolysis is released upon AUG selection by the 40S preinitiation complex. These results, taken together, demonstrate the biochemical requirements for regulation of GTP hydrolysis and its coupling to the AUG selection process during translation initiation.     

Interaction of liver plasma membranes and GTP with GTP hydrolysis

[¹⁴C]GTP or a metabolic product of GTP binds to liver membranes. Less label was associated with membranes when membranes were incubated with increasing concentrations of carrier GTP; ATP did not displace the label. Chromatography of extracted incubation mixtures of [¹⁴C]GTP and membranes revealed that over 96% of the nucleotide was hydrolyzed to 5′GMP and guanosine, Exposure of liver membranes to GTP prevented the separation of characteristic membrane bands that could be obtained when centrifugation was carried out without GTP. These studies indicate that GTP-effected alteration of liver plasma membranes is concomitant with GTP hydrolysis. These effects may be in addition to direct effects of GTP on enzymes and membrane proteins.     

GTP hydrolysis mechanism of Ras-like GTPases

The Ras-like GTPases regulate diverse cellular functions via the chemical cycle of binding and hydrolyzing GTP molecules. They alternate between GTP- and GDP-bound conformations. The GTP-bound conformation is biologically active and promotes a cellular function, such as signal transduction, cytoskeleton organization, protein synthesis/translocation, or a membrane budding/fusion event. GTP hydrolysis turns off the GTPase switch by converting it to the inactive GDP-bound conformation. The fundamental GTP hydrolysis mechanism by these GTPases has generated considerable interest over the last two decades but remained to be firmly established. This review provides an update on the catalytic mechanism with discussions on recent developments from kinetic, structural, and model studies in the context of the various GTP hydrolysis models proposed over the years.     

Protein-lipid interactions in membrane trafficking at the Golgi complex

The integrated interplay between proteins and lipids drives many key cellular processes, such as signal transduction, cytoskeleton remodelling and membrane trafficking. The last of these, membrane trafficking, has the Golgi complex as its central station. Not only does this organelle orchestrates the biosynthesis, transport and intracellular distribution of many proteins and lipids, but also its own function and structure is dictated by intimate functional and physical relationships between protein-based and lipid-based machineries. These machineries are involved in the control of the fundamental events that govern membrane traffic, such as in the budding, fission and fusion of transport intermediates, in the regulation of the shape and geometry of the Golgi membranes themselves, and, finally, in the generation of "signals" that can have local actions in the secretory system, or that may affect other cellular systems. Lipid-protein interactions rely on the abilities of certain protein domains to recognize specific lipids. These interactions are mediated, in particular, through the headgroups of the phospholipids, although a few of these protein domains are able to specifically interact with the phospholipid acyl chains. Recent evidence also indicates that some proteins and/or protein domains are more sensitive to the physical environment of the membrane bilayer (such as its curvature) than to its chemical composition.     

Lipid-transfer proteins in membrane trafficking at the Golgi complex

The Golgi complex (GC) represents the central junction for membrane trafficking. Protein and lipid cargoes continuously move through the GC in both anterograde and retrograde directions, departing to and arriving from diverse destinations within the cell. Nevertheless, the GC is able to maintain its identity and strict compartmentalisation, having a different composition in terms of protein and lipid content compared to other organelles. The discovery of coat protein complexes and the elucidation of their role in sorting cargo proteins into specific transport carriers have provided a partial answer to this phenomenon. However, it is more difficult to understand how relatively small and diffusible molecules like lipids can be concentrated in or excluded from specific subcellular compartments. The discovery of lipid-transfer proteins operating in the secretory pathway and specifically at the GC has shed light on one possible way in which this lipid compartmentalisation can be accomplished. The correct lipid distribution along the secretory pathway is of crucial importance for cargo protein sorting and secretion. This review focuses on what is now known about the putative and effective lipid-transfer proteins at the GC, and on how they affect the function and structure of the GC itself.     

Effectors increase the affinity of ADP-ribosylation factor for GTP to increase binding

The stoichiometry of the binding of GTP to ADP-ribosylation factor (ARF) proteins, normally quite low at approximately 0.05 mol/mol protein, was found to increase to a maximum of 1 mol/mol in the presence of effectors. The mechanism of this action was found to result from the ability of these effectors to increase the affinity of ARF for activating guanine nucleotide triphosphates. The existence of a conformation of ARF with low affinity (>100 micrometer) for GTP is proposed. The actions of effectors to increase the equilibrium binding of GTP is interpreted as evidence that these same effectors interact with and modulate the affinity of the inactive ARF for GTP. A new model for these interactions among ARF, effectors, and GTP is proposed, and a preliminary test in cells is supportive of these observations with relevance to signaling in cells.     

Effects of inhibitors of tubulin polymerization on GTP hydrolysis

The effects of a number of antimitotic drugs on the GTPase activity of tubulin were examined. The previously reported stimulation with colchicine and inhibition with podophyllotoxin and vinblastine wee confirmed. Maytansine, which competes with vinblastine in binding to tubulin, was comparable to the latter in inhibiting GTP hydrolysis. Nocodazole, which competes with colchicine in binding to tubulin, was significantly superior to colchicine in enhancing GTP hydrolysis. This superiority arose from the more rapid bindng of nocodazole to tubulin, as the two drugs had comparable activity when drug and tubulin were preincubated prior to the addition of GTP. Both colchicine and podophyllotoxin contain a trimethoxybenzene ring, while the closest structural analogy of nocodazole to colchicine includes the trimethoxybenzene ring. To explore this apparent paradox, we examined a number of simpler colchicine analogs for their effects on tubulin-dependent GTP hydrolysis. While tropolone was without effect, 3,4,5-trimethoxybenzaldehyde and 2,3,4-trimethoxybenzaldehyde stimulated the reaction. We therefore conclude that the trimethoxybenzene ring of colchicine is primarily responsible for the drug's stimulation of the GTPase activity of tubulin and that the inhibitory effect of podophyllotoxin must derive from the latter's tetrahydronaphthol moiety.     

Different ubiquitin signals act at the Golgi and plasma membrane to direct GAP1 trafficking

The high capacity general amino acid permease, Gap1p, in Saccharomyces cerevisiae is distributed between the plasma membrane and internal compartments according to availability of amino acids. When internal amino acid levels are low, Gap1p is localized to the plasma membrane where it imports available amino acids from the medium. When sufficient amino acids are imported, Gap1p at the plasma membrane is endocytosed and newly synthesized Gap1p is delivered to the vacuole; both sorting steps require Gap1p ubiquitination. Although it has been suggested that identical trans-acting factors and Gap1p ubiquitin acceptor sites are involved in both processes, we define unique requirements for each of the ubiquitin-mediated sorting steps involved in delivery of Gap1p to the vacuole upon amino acid addition. Our finding that distinct ubiquitin-mediated sorting steps employ unique trans-acting factors, ubiquitination sites on Gap1p, and types of ubiquitination demonstrates a previously unrecognized level of specificity in ubiquitin-mediated protein sorting.     

ADP-ribosylation of histones of the chicken liver nucleus at different rates of glycohydrolase hydrolysis of NAD

The rate of incorporation of nicotinamide-[adenosine-U-14C]adenine dinucleotide [( Ado-U-14C]NAD) into histones and the poly(ADPR) polymerase activity of chromatin suggest that the NAD-dependent ADP-ribosylation of histones depends on the rate of NAD hydrolysis by glycohydrolase in chicken liver nuclei. With a rise in the NAD-glycohydrolase activity after treatment of nuclei with Triton X-100 the synthesis of poly(ADP-ribose) via the poly(ADPR)polymerase reaction is augmented, as a result of which the rate of [Ado-U-14C]NAD incorporation into total histones is increased. On the contrary, the decrease of NAD-glycohydrolase hydrolysis after treatment of nuclei with SDS lowers the poly(ADPR)polymerase activity and [Ado-U-14C]NAD incorporation into histones. Under these conditions, i. e. different rates of glycohydrolase hydrolysis of NAD in the nuclei, some redistribution of [Ado U-14C]NAD incorporation into individual histones occurs.     

Real-time in vitro measurement of GTP hydrolysis

Small GTPases require an active GTPase activity to function correctly in their cellular environment. Mutation of key residues involved in this activity renders the GTPase defective and the small G-protein constitutively active (GTP-locked). The GTPase activity is also a target for GTPase-activating proteins (GAPs) which act to attenuate GTPase signalling by accelerating the conversion of bound GTP to bound GDP. The measurement of GTP hydrolysis in vitro can therefore provide information on the intrinsic activity of the small GTPase (e.g., mutated GTPase activity) as well as help define GAP specificity. Current methods to measure GTP hydrolysis in vitro utilise either radioactivity-based filter-binding assays or measurements of GDP:GTP:P(i) ratios by high-performance liquid chromatography (HPLC). Both provide timed snapshots of the current GTP-bound state, can be prone to experimental errors, and do not provide a real-time observation of GTP hydrolysis. The method we describe here utilises a fluorescently labelled, phosphate-binding protein (PBP), which scavenges for free inorganic phosphate (P(i)). On binding of a single P(i), a change of protein conformation is coupled to a 7-fold increase in fluorescence of the fluorophore. This method therefore permits real-time monitoring of GTPase activity, through measurement of P(i) production. This review describes the process of preparing and labelling the PBP with the MDCC fluorophore, as well as an example of its use in measuring the GTPase activity of small GTPases. We also discuss the pros and cons, and implications of the technique in comparison to the radioactive and HPLC method of measuring the GTPase activity.     

Regulation of acetyl-CoA carboxylase by ADP-ribosylation

Because of certain similarities between acetyl-CoA carboxylase (ACC) and tubulin, and the recent demonstration of the ADP-ribosylation of tubulin by cholera toxin, we have investigated a potential role for ADP-ribosylation in the regulation of ACC activity. Incubation of purified rat liver ACC with cholera toxin in the presence of millimolar concentrations of [adenylate-32P]NAD results in a time-dependent incorporation of ADP-ribose into ACC of greater than 2 mol/mol of enzyme subunit, accompanied by a marked inactivation of enzyme activity. This effect is not mimicked by pertussis toxin, ADP-ribose, or ribose 5-phosphate. Incubation of labeled ACC with snake venom phosphodiesterase and alkaline hydrolysis release 32P-products tentatively identified by high-performance liquid chromatography as 5'-[32P]AMP and [32P]ADP-ribose, respectively. These data are consistent with a mono-ADP-ribosylation of ACC catalyzed by cholera toxin. Phosphodiesterase treatment of inactivated ACC partially restores enzyme activity. The effects of ADP-ribosylation of ACC are expressed both as a decrease in the enzyme Vmax and as an increase in the apparent Ka for citrate. These results suggest that ACC might be a substrate for endogenous ADP-ribosyltransferases and that this covalent modification could be an important regulatory mechanism for the modulation of fatty acid synthesis in vivo.     

Recruitment to Golgi membranes of ADP-ribosylation factor 1 is mediated by the cytoplasmic domain of p23.

Binding to Golgi membranes of ADP ribosylation factor 1 (ARF1) is the first event in the initiation of COPI coat assembly. Based on binding studies, a proteinaceous receptor has been proposed to be critical for this process. We now report that p23, a member of the p24 family of Golgi-resident transmembrane proteins, is involved in ARF1 binding to membranes. Using a cross-link approach based on a photolabile peptide corresponding to the cytoplasmic domain of p23, the GDP form of ARF1 (ARF1-GDP) is shown to interact with p23 whereas ARF1-GTP has no detectable affinity to p23. The p23 binding is shown to localize specifically to a 22 amino acid C-terminal fragment of ARF1. While a monomeric form of a non-photolabile p23 peptide does not significantly inhibit formation of the cross-link product, the corresponding dimeric form does compete efficiently for this interaction. Consistently, the dimeric p23 peptide strongly inhibits ARF1 binding to native Golgi membranes suggesting that an oligomeric form of p23 acts as a receptor for ARF1 before nucleotide exchange takes place.