Calcium release from the endoplasmic reticulum of higher plants elicited by the NADP metabolite nicotinic acid adenine dinucleotide phosphate

Higher plants share with animals a responsiveness to the Ca2+ mobilizing agents inositol 1,4,5-trisphosphate (InsP3) and cyclic ADP-ribose (cADPR). In this study, by using a vesicular 45Ca2+ flux assay, we demonstrate that microsomal vesicles from red beet and cauliflower also respond to nicotinic acid adenine dinucleotide phosphate (NAADP), a Ca2+-releasing molecule recently described in marine invertebrates. NAADP potently mobilizes Ca2+ with a K1/2 = 96 nM from microsomes of nonvacuolar origin in red beet. Analysis of sucrose gradient-separated cauliflower microsomes revealed that the NAADP-sensitive Ca2+ pool was derived from the endoplasmic reticulum. This exclusively nonvacuolar location of the NAADP-sensitive Ca2+ pathway distinguishes it from the InsP3- and cADPR-gated pathways. Desensitization experiments revealed that homogenates derived from cauliflower tissue contained low levels of NAADP (125 pmol/mg) and were competent in NAADP synthesis when provided with the substrates NADP and nicotinic acid. NAADP-induced Ca2+ release is insensitive to heparin and 8-NH2-cADPR, specific inhibitors of the InsP3- and cADPR-controlled mechanisms, respectively. However, NAADP-induced Ca2+ release could be blocked by pretreatment with a subthreshold dose of NAADP, as previously observed in sea urchin eggs. Furthermore, the NAADP-gated Ca2+ release pathway is independent of cytosolic free Ca2+ and therefore incapable of operating Ca2+-induced Ca2+ release. In contrast to the sea urchin system, the NAADP-gated Ca2+ release pathway in plants is not blocked by L-type channel antagonists. The existence of multiple Ca2+ mobilization pathways and Ca2+ release sites might contribute to the generation of stimulus-specific Ca2+ signals in plant cells.

Adenosine diphosphate glucose pyrophosphatase: A plastidial phosphodiesterase that prevents starch biosynthesis

A distinct phosphodiesterasic activity (EC 3.1.4) was found in both mono- and dicotyledonous plants that catalyzes the hydrolytic breakdown of ADPglucose (ADPG) to produce equimolar amounts of glucose-1-phosphate and AMP. The enzyme responsible for this activity, referred to as ADPG pyrophosphatase (AGPPase), was purified over 1,100-fold from barley leaves and subjected to biochemical characterization. The calculated Keq′ (modified equilibrium constant) value for the ADPG hydrolytic reaction at pH 7.0 and 25°C is 110, and its standard-state free-energy change value (ΔG′) is −2.9 kcal/mol (1 kcal = 4.18 kJ). Kinetic analyses showed that, although AGPPase can hydrolyze several low-molecular weight phosphodiester bond-containing compounds, ADPG proved to be the best substrate (Km = 0.5 mM). Pi and phosphorylated compounds such as 3-phosphoglycerate, PPi, ATP, ADP, NADP+, and AMP are inhibitors of AGPPase. Subcellular localization studies revealed that AGPPase is localized exclusively in the plastidial compartment of cultured cells of sycamore (Acer pseudoplatanus L.), whereas it occurs both inside and outside the plastid in barley endosperm. In this paper, evidence is presented that shows that AGPPase, whose activity declines concomitantly with the accumulation of starch during development of sink organs, competes with starch synthase (ADPG:1,4-α-d-glucan 4-α-d-glucosyltransferase; EC 2.4.1.21) for ADPG, thus markedly blocking the starch biosynthesis.

High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia

Mitochondria are confronted with low oxygen levels in the microenvironment within tissues; yet, isolated mitochondria are routinely studied under air-saturated conditions that are effectively hyperoxic, increase oxidative stress, and may impair mitochondrial function. Under hypoxia, on the other hand, respiration and ATP supply are restricted. Under these conditions of oxygen limitation, any compromise in the coupling of oxidative phosphorylation to oxygen consumption could accentuate ATP depletion, leading to metabolic failure. To address this issue, we have developed the approach of oxygen-injection microcalorimetry and ADP-injection respirometry for evaluating mitochondrial function at limiting oxygen supply. Whereas phosphorylation efficiency drops during ADP limitation at high oxygen levels, we show here that oxidative phosphorylation is more efficient at low oxygen than at air saturation, as indicated by higher ratios of ADP flux to total oxygen flux at identical submaximal rates of ATP synthesis. At low oxygen, the proton leak and uncoupled respiration are depressed, thus reducing maintenance energy expenditure. This indicates the importance of low intracellular oxygen levels in avoiding oxidative stress and protecting bioenergetic efficiency.

Rubisco activase constrains the photosynthetic potential of leaves at high temperature and CO2

Net photosynthesis (Pn) is inhibited by moderate heat stress. To elucidate the mechanism of inhibition, we examined the effects of temperature on gas exchange and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activation in cotton and tobacco leaves and compared the responses to those of the isolated enzymes. Depending on the CO2 concentration, Pn decreased when temperatures exceeded 35–40°C. This response was inconsistent with the response predicted from the properties of fully activated Rubisco. Rubisco deactivated in leaves when temperature was increased and also in response to high CO2 or low O2. The decrease in Rubisco activation occurred when leaf temperatures exceeded 35°C, whereas the activities of isolated activase and Rubisco were highest at 42°C and >50°C, respectively. In the absence of activase, isolated Rubisco deactivated under catalytic conditions and the rate of deactivation increased with temperature but not with CO2. The ability of activase to maintain or promote Rubisco activation in vitro also decreased with temperature but was not affected by CO2. Increasing the activase/Rubisco ratio reduced Rubisco deactivation at higher temperatures. The results indicate that, as temperature increases, the rate of Rubisco deactivation exceeds the capacity of activase to promote activation. The decrease in Rubisco activation that occurred in leaves at high CO2 was not caused by a faster rate of deactivation, but by reduced activase activity possibly in response to unfavorable ATP/ADP ratios. When adjustments were made for changes in activation state, the kinetic properties of Rubisco predicted the response of Pn at high temperature and CO2.

The domain structure of protoporphyrinogen oxidase, the molecular target of diphenyl ether-type herbicides

Protoporphyrinogen oxidase (EC 1–3-3–4), the 60-kDa membrane-bound flavoenzyme that catalyzes the final reaction of the common branch of the heme and chlorophyll biosynthesis pathways in plants, is the molecular target of diphenyl ether-type herbicides. It is highly resistant to proteases (trypsin, endoproteinase Glu-C, or carboxypeptidases A, B, and Y), because the protein is folded into an extremely compact form. Trypsin maps of the native purified and membrane-bound yeast protoporphyrinogen oxidase show that this basic enzyme (pI > 8.5) was cleaved at a single site under nondenaturing conditions, generating two peptides with relative molecular masses of 30,000 and 35,000. The endoproteinase Glu-C also cleaved the protein into two peptides with similar masses, and there was no additional cleavage site under mild denaturing conditions. N-terminal peptide sequence analysis of the proteolytic (trypsin and endoproteinase Glu-C) peptides showed that both cleavage sites were located in putative connecting loop between the N-terminal domain (25 kDa) with the βαβ ADP-binding fold and the C-terminal domain (35 kDa), which possibly is involved in the binding of the isoalloxazine moiety of the FAD cofactor. The peptides remained strongly associated and fully active with the Km for protoporphyrinogen and the Ki for various inhibitors, diphenyl-ethers, or diphenyleneiodonium derivatives, identical to those measured for the native enzyme. However, the enzyme activity of the peptides was much more susceptible to thermal denaturation than that of the native protein. Only the C-terminal domain of protoporphyrinogen oxidase was labeled specifically in active site-directed photoaffinity-labeling experiments. Trypsin may have caused intramolecular transfer of the labeled group to reactive components of the N-terminal domain, resulting in nonspecific labeling. We suggest that the active site of protoporphyrinogen oxidase is in the C-terminal domain of the protein, at the interface between the C- and N-terminal domains.

A molecular mechanism for signaling between seven-transmembrane receptors: evidence for a redistribution of G proteins

Although activation of one seven-transmembrane receptor can influence the response of a separate seven-transmembrane receptor, e.g., the phenomenon of synergism, the underlying mechanism(s) for this signaling process is unclear. The present study investigated communication between two receptors that exhibit classical synergism, e.g., human platelet thrombin and thromboxane A2 receptors. Activation of thrombin receptors caused an increase in ligand affinity of thromboxane A2 receptors. This effect (i) was shown to be specific, since a similar increase in ligand affinity was not caused by ADP or A23187; (ii) did not require cytosolic components, e.g., kinases, proteases, phosphatases, etc., because it occurred in isolated platelet membranes; (iii) was G protein-mediated because it was blocked by an Gαq C terminus antibody; and (iv) was associated with a net increase in Gαq coupling to thromboxane A2 receptors. Collectively, these data provide evidence that seven-transmembrane receptors that share a common Gα subunit can communicate with each other via a redistribution of their G proteins. Thus, activation of thrombin receptors increases Gαq association with thromboxane A2 receptors thereby shifting them to a higher affinity state. This signaling phenomenon, which modulates receptor-ligand affinity, may serve as a molecular mechanism for cellular adaptive processes such as synergism.

Minimal and optimal mechanisms for GroE-mediated protein folding

We have analyzed the effects of different components of the GroE chaperonin system on protein folding by using a nonpermissive substrate (i.e., one that has very low spontaneous refolding yield) for which rate data can be acquired. In the absence of GroES and nucleotides, the rate of GroEL-mediated refolding of heat- and DTT-denatured mitochondrial malate dehydrogenase was extremely low, but some three times higher than the spontaneous rate. This GroEL-mediated rate was increased 17-fold by saturating concentrations of ATP, 11-fold by ADP and GroES, and 465-fold by ATP and GroES. Optimal refolding activity was observed when the dissociation of GroES from the chaperonin complex was dramatically reduced. Although GroEL minichaperones were able to bind denatured mitochondrial malate dehydrogenase, they were ineffective in enhancing the refolding rate. The spectrum of mechanisms for GroE-mediated protein folding depends on the nature of the substrate. The minimal mechanism for permissive substrates (i.e., having significant yields of spontaneous refolding), requires only binding to the apical domain of GroEL. Slow folding rates of nonpermissive substrates are limited by the transitions between high- and low-affinity states of GroEL alone. The optimal mechanism, which requires holoGroEL, physiological amounts of GroES, and ATP hydrolysis, is necessary for the chaperonin-mediated folding of nonpermissive substrates at physiologically relevant rates under conditions in which retention of bound GroES prevents the premature release of aggregation-prone folding intermediates from the chaperonin complex. The different mechanisms are described in terms of the structural features of mini- and holo-chaperones.

Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: The effect of nucleotides and actin

Myosin is thought to generate movement of actin filaments via a conformational change between its light-chain domain and its catalytic domain that is driven by the binding of nucleotides and actin. To monitor this change, we have measured distances between a gizzard regulatory light chain (Cys 108) and the active site (near or at Trp 130) of skeletal myosin subfragment 1 (S1) by using luminescence resonance energy transfer and a photoaffinity ATP-lanthanide analog. The technique allows relatively long distances to be measured, and the label enables site-specific attachment at the active-site with only modest affect on myosin’s enzymology. The distance between these sites is 66.8 ± 2.3 Å when the nucleotide is ADP and is unchanged on binding to actin. The distance decreases slightly with ADP-BeF3, (−1.6 ± 0.3 Å) and more significantly with ADP-AlF4 (−4.6 ± 0.2 Å). During steady-state hydrolysis of ATP, the distance is temperature-dependent, becoming shorter as temperature increases and the complex with ADP⋅Pi is favored over that with ATP. We conclude that the distance between the active site and the light chain varies as Acto-S1-ADP ≈ S1-ADP > S1-ADP-BeF3 > S1-ADP-AlF4 ≈ S1-ADP-Pi and that S1-ATP > S1-ADP-Pi. The changes in distance are consistent with a substantial rotation of the light-chain binding domain of skeletal S1 between the prepowerstroke state, simulated by S1-ADP-AlF4, and the post-powerstroke state, simulated by acto-S1-ADP.

Uncoupling proteins 2 and 3 are highly active H+ transporters and highly nucleotide sensitive when activated by coenzyme Q (ubiquinone)

Based on the discovery of coenzyme Q (CoQ) as an obligatory cofactor for H+ transport by uncoupling protein 1 (UCP1) [Echtay, K. S., Winkler, E. & Klingenberg, M. (2000) Nature (London) 408, 609–613] we show here that UCP2 and UCP3 are also highly active H+ transporters and require CoQ and fatty acid for H+ transport, which is inhibited by low concentrations of nucleotides. CoQ is proposed to facilitate injection of H+ from fatty acid into UCP. Human UCP2 and 3 expressed in Escherichia coli inclusion bodies are solubilized, and by exchange of sarcosyl against digitonin, nucleotide binding as measured with 2′-O-[5-(dimethylamino)naphthalene-1-sulfonyl]-GTP can be restored. After reconstitution into vesicles, Cl− but no H+ are transported. The addition of CoQ initiates H+ transport in conjunction with fatty acids. This increase is fully sensitive to nucleotides. The rates are as high as with reconstituted UCP1 from mitochondria. Maximum activity is at a molar ratio of 1:300 of CoQ:phospholipid. In UCP2 as in UCP1, ATP is a stronger inhibitor than ADP, but in UCP3 ADP inhibits more strongly than ATP. Thus UCP2 and UCP3 are regulated differently by nucleotides, in line with their different physiological contexts. These results confirm the regulation of UCP2 and UCP3 by the same factors CoQ, fatty acids, and nucleotides as UCP1. They supersede reports that UCP2 and UCP3 may not be H+ transporters.

Trapping the transition state of an ATP-binding cassette transporter: Evidence for a concerted mechanism of maltose transport

High-affinity uptake into bacterial cells is mediated by a large class of periplasmic binding protein-dependent transport systems, members of the ATP-binding cassette superfamily. In the maltose transport system of Escherichia coli, the periplasmic maltose-binding protein binds its substrate maltose with high affinity and, in addition, stimulates the ATPase activity of the membrane-associated transporter when maltose is present. Vanadate inhibits maltose transport by trapping ADP in one of the two nucleotide-binding sites of the membrane transporter immediately after ATP hydrolysis, consistent with its ability to mimic the transition state of the γ-phosphate of ATP during hydrolysis. Here we report that the maltose-binding protein becomes tightly associated with the membrane transporter in the presence of vanadate and simultaneously loses its high affinity for maltose. These results suggest a general model explaining how ATP hydrolysis is coupled to substrate transport in which a binding protein stimulates the ATPase activity of its cognate transporter by stabilizing the transition state.

A thrombin receptor function for platelet glycoprotein Ib–IX unmasked by cleavage of glycoprotein V

Glycoprotein (GP) V is a major substrate cleaved by the protease thrombin during thrombin-induced platelet activation. Previous analysis of platelets from GP V-null mice suggested a role for GP V as a negative modulator of platelet activation by thrombin. We now report the mechanism by which thrombin activates GP V −/− platelets. We show that proteolytically inactive forms of thrombin induce robust stimulatory responses in GP V null mouse platelets, via the platelet GP Ib–IX–V complex. Because proteolytically inactive thrombin can activate wild-type mouse and human platelets after treatment with thrombin to cleave GP V, this mechanism is involved in thrombin-induced platelet aggregation. Platelet activation through GP Ib–IX depends on ADP secretion, and specific inhibitors demonstrate that the recently cloned P2Y12 ADP receptor (Gi-coupled ADP receptor) is involved in this pathway, and that the P2Y1 receptor (Gq-coupled ADP receptor) may play a less significant role. Thrombosis was generated in GP V null mice only in response to catalytically inactive thrombin, whereas thrombosis occurred in both genotypes (wild type and GP V null) in response to active thrombin. These data support a thrombin receptor function for the platelet membrane GP Ib–IX–V complex, and describe a novel thrombin signaling mechanism involving an initiating proteolytic event followed by stimulation of the GP Ib–IX via thrombin acting as a ligand, resulting in platelet activation.

Characterization of Schizosaccharomyces pombe RNA triphosphatase

RNA triphosphatase catalyzes the first step in mRNA cap formation which entails the cleavage of the β–γ phosphoanhydride bond of triphosphate-terminated RNA to yield a diphosphate end that is then capped with GMP by RNA guanylyltransferase. Here we characterize a 303 amino acid RNA triphosphatase (Pct1p) encoded by the fission yeast Schizosaccharomyces pombe. Pct1p hydrolyzes the γ phosphate of triphosphate-terminated poly(A) in the presence of magnesium. Pct1p also hydrolyzes ATP to ADP and Pi in the presence of manganese or cobalt (Km = 19 µM ATP; kcat = 67 s–1). Hydrolysis of 1 mM ATP is inhibited with increasing potency by inorganic phosphate (I0.5 = 1 mM), pyrophosphate (I0.5 = 0.4 mM) and tripolyphosphate (I0.5 = 30 µM). Velocity sedimentation indicates that Pct1p is a homodimer. Pct1p is biochemically and structurally similar to the catalytic domain of Saccharomyces cerevisiae RNA triphosphatase Cet1p. Mechanistic conservation between Pct1p and Cet1p is underscored by a mutational analysis of the putative metal-binding site of Pct1p. Pct1p is functional in vivo in S.cerevisiae in lieu of Cet1p, provided that it is coexpressed with the S.pombe guanylyltransferase. Pct1p and other yeast RNA triphosphatases are completely unrelated, mechanistically and structurally, to the metazoan RNA triphosphatases, suggesting an abrupt evolutionary divergence of the capping apparatus during the transition from fungal to metazoan species.

The human mitochondrial deoxynucleotide carrier and its role in the toxicity of nucleoside antivirals

The synthesis of DNA in mitochondria requires the uptake of deoxynucleotides into the matrix of the organelle. We have characterized a human cDNA encoding a member of the family of mitochondrial carriers. The protein has been overexpressed in bacteria and reconstituted into phospholipid vesicles where it catalyzed the transport of all four deoxy (d) NDPs, and, less efficiently, the corresponding dNTPs, in exchange for dNDPs, ADP, or ATP. It did not transport dNMPs, NMPs, deoxynucleosides, nucleosides, purines, or pyrimidines. The physiological role of this deoxynucleotide carrier is probably to supply deoxynucleotides to the mitochondrial matrix for conversion to triphosphates and incorporation into mitochondrial DNA. The protein is expressed in all human tissues that were examined except for placenta, in accord with such a central role. The deoxynucleotide carrier also transports dideoxynucleotides efficiently. It is likely to be medically important by providing the means of uptake into mitochondria of nucleoside analogs, leading to the mitochondrial impairment that underlies the toxic side effects of such drugs in the treatment of viral illnesses, including AIDS, and in cancer therapy.

Multiple Roles of Arf1 GTPase in the Yeast Exocytic and Endocytic Pathways

ADP-ribosylation factors, a family of small GTPases, are believed to be key regulators of intracellular membrane traffic. However, many biochemical in vitro experiments have led to different models for their involvement in various steps of vesicular transport, and their precise role in living cells is still unclear. We have taken advantage of the powerful yeast genetic system and screened for temperature-sensitive (ts) mutants of the ARF1 gene from Saccharomyces cerevisiae. By random mutagenesis of the whole open reading frame of ARF1 by error-prone PCR, we isolated eight mutants and examined their phenotypes. arf1 ts mutants showed a variety of transport defects and morphological alterations in an allele-specific manner. Furthermore, intragenic complementation was observed between certain pairs of mutant alleles, both for cell growth and intracellular transport. These results demonstrate that the single Arf1 protein is indeed involved in many different steps of intracellular transport in vivo and that its multiple roles may be dissected by the mutant alleles we constructed.

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 105–106 molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca2+-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.