The medial-Golgi Ion Pump Pmr1 Supplies the Yeast Secretory Pathway with Ca2+ and Mn2+ Required for Glycosylation, Sorting, and Endoplasmic Reticulum-Associated Protein Degradation

The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.

Membrane voltage initiates Ca2+ waves and potentiates Ca2+ increases with abscisic acid in stomatal guard cells

In higher plants changes and oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) are central to hormonal physiology, including that of abscisic acid (ABA), which signals conditions of water stress and alters ion channel activities in guard cells of higher-plant leaves. Such changes in [Ca2+]i are thought to encode for cellular responses to different stimuli, but their origins and functions are poorly understood. Because transients and oscillations in membrane voltage also occur in guard cells and are elicited by hormones, including ABA, we suspected a coupling of [Ca2+]i to voltage and its interaction with ABA. We recorded [Ca2+]i by Fura2 fluorescence ratio imaging and photometry while bringing membrane voltage under experimental control with a two-electrode voltage clamp in intact Vicia guard cells. Free-running oscillations between voltages near ���50 mV and ���200 mV were associated with oscillations in [Ca2+]i, and, under voltage clamp, equivalent membrane hyperpolarizations caused [Ca2+]i to increase, often in excess of 1 ��M, from resting values near 100 nM. Image analysis showed that the voltage stimulus evoked a wave of high [Ca2+]i that spread centripetally from the peripheral cytoplasm within 5���10 s and relaxed over 40���60 s thereafter. The [Ca2+]i increases showed a voltage threshold near ���120 mV and were sensitive to external Ca2+ concentration. Substituting Mn2+ for Ca2+ to quench Fura2 fluorescence showed that membrane hyperpolarization triggered a divalent influx. ABA affected the voltage threshold for the [Ca2+]i rise, its amplitude, and its duration. In turn, membrane voltage determined the ability of ABA to raise [Ca2+]i. These results demonstrate a capacity for voltage to evoke [Ca2+]i increases, they point to a dual interaction with ABA in triggering and propagating [Ca2+]i increases, and they implicate a role for voltage in ���conditioning��� [Ca2+]i signals that regulate ion channels for stomatal function.

Tn552 transposase catalyzes concerted strand transfer in vitro

The Tn552 transposase, a member of the DDE superfamily of transposase and retroviral integrase proteins, has been expressed in soluble form. The purified protein performs concerted strand transfer in vitro, efficiently pairing two preprocessed transposon ends and inserting them into target DNA. For maximum efficiency, both participating DNA ends must contain the two adjacent transposase-binding sites that are the normal constituents of the Tn552 termini. As is the case with transposition in vivo, the insertions recovered from the reaction in vitro are flanked by repeats of a short target sequence, most frequently 6 bp. The reaction has stringent requirements for a divalent metal ion. Concerted strand transfer is most efficient with Mg2+. Although it stimulates strand transfer overall, Mn2+ promotes uncoupled, single-ended events at the expense of concerted insertions. The simplicity and efficiency of the Tn552 transposition system make it an attractive subject for structural and biochemical studies and a potentially useful genetic tool.

Histidine kinase activity of the ETR1 ethylene receptor from Arabidopsis

ETR1 represents a prototypical ethylene receptor. Homologues of ETR1 have been identified in Arabidopsis as well as in other plant species, indicating that ethylene perception involves a family of receptors and that the mechanism of ethylene perception is conserved in plants. The amino-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization. The carboxyl-terminal half of the polypeptide contains domains with homology to histidine kinases and response regulators, signaling motifs originally identified in bacteria. The putative histidine kinase domain of ETR1 was expressed in yeast as a fusion protein with glutathione S-transferase and affinity purified. Autophosphorylation of the purified fusion protein was observed on incubation with radiolabeled ATP. The incorporated phosphate was resistant to treatment with 3 M NaOH, but was sensitive to 1 M HCl, consistent with phosphorylation of histidine. Autophosphorylation was abolished by mutations that eliminated either the presumptive site of phosphorylation (His-353) or putative catalytic residues within the kinase domain. Truncations were used to delineate the region required for histidine kinase activity. An examination of cation requirements indicated that ETR1 requires Mn2+ for autophosphorylation. These results demonstrate that higher plants contain proteins with histidine kinase activity. Furthermore, these results indicate that aspects of ethylene signaling may be regulated by changes in histidine kinase activity of the receptor.

ECA1 complements yeast mutants defective in Ca2+ pumps and encodes an endoplasmic reticulum-type Ca2+-ATPase in Arabidopsis���thaliana

To understand the structure, role, and regulation of individual Ca2+ pumps in plants, we have used yeast as a heterologous expression system to test the function of a gene from Arabidopsis thaliana (ECA1). ECA1 encoded a 116-kDa polypeptide that has all the conserved domains common to P-type Ca2+ pumps (EC 3.6.1.38). The amino acid sequence shared more identity with sarcoplasmic/endoplasmic reticulum (53%) than with plasma membrane (32%) Ca2+ pumps. Yeast mutants defective in a Golgi Ca2+ pump (pmr1) or both Golgi and vacuolar Ca2+ pumps (pmr1 pmc1 cnb1) were sensitive to growth on medium containing 10 mM EGTA or 3 mM Mn2+. Expression of ECA1 restored growth of either mutant on EGTA. Membranes were isolated from the pmr1 pmc1 cnb1 mutant transformed with ECA1 to determine if the ECA1 polypeptide (ECA1p) could be phosphorylated as intermediates of the reaction cycle of Ca2+-pumping ATPases. In the presence of [��-32P]ATP, ECA1p formed a Ca2+-dependent [32P]phosphoprotein of 106 kDa that was sensitive to hydroxylamine. Cyclopiazonic acid, a blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+ pumps, inhibited the formation of the phosphoprotein, whereas thapsigargin did not. Immunoblotting with an antibody against the carboxyl tail showed that ECA1p was associated mainly with the endoplasmic reticulum membranes isolated from Arabidopsis plants. The results support the model that ECA1 encodes an endoplasmic reticulum-type Ca2+ pump in Arabidopsis. The ability of ECA1p to restore growth of mutant pmr1 on medium containing Mn2+, and the formation of a Mn2+-dependent phosphoprotein suggested that ECA1p may also regulate Mn2+ homeostasis by pumping Mn2+ into endomembrane compartments of plants.

A curved RNA helix incorporating an internal loop with G��A and A��A non-Watson���Crick���base���pairing

The crystal structure of the RNA dodecamer 5���-GGCC(GAAA)GGCC-3��� has been determined from x-ray diffraction data to 2.3-��� resolution. In the crystal, these oligomers form double helices around twofold symmetry axes. Four consecutive non-Watson���Crick base pairs make up an internal loop in the middle of the duplex, including sheared G��A pairs and novel asymmetric A��A pairs. This internal loop sequence produces a significant curvature and narrowing of the double helix. The helix is curved by 34�� from end to end and the diameter is narrowed by 24% in the internal loop. A Mn2+ ion is bound directly to the N7 of the first guanine in the Watson���Crick region following the internal loop and the phosphate of the preceding residue. This Mn2+ location corresponds to a metal binding site observed in the hammerhead catalytic RNA.

Isolation and bacterial expression of a sesquiterpene synthase cDNA clone from peppermint (Mentha x piperita, L.) that produces the aphid alarm pheromone���(E)-��-farnesene

(E)-��-Farnesene is a sesquiterpene semiochemical that is used extensively by both plants and insects for communication. This acyclic olefin is found in the essential oil of peppermint (Mentha x piperita) and can be synthesized from farnesyl diphosphate by a cell-free extract of peppermint secretory gland cells. A cDNA from peppermint encoding (E)-��-farnesene synthase was cloned by random sequencing of an oil gland library and was expressed in Escherichia coli. The corresponding synthase has a deduced size of 63.8 kDa and requires a divalent cation for catalysis (Km for Mg2+ ��� 150 ��M; Km for Mn2+ ��� 7 ��M). The sesquiterpenoids produced by the recombinant enzyme, as determined by radio-GC and GC-MS, are (E)-��-farnesene (85%), (Z)-��-farnesene (8%), and ��-cadinene (5%) with the native C15 substrate farnesyl diphosphate (Km ��� 0.6 ��M; Vrel = 100) and Mg2+ as cofactor, and (E)-��-farnesene (98%) and (Z)-��-farnesene (2%) with Mn2+ as cofactor (Vrel = 80). With the C10 analog, GDP, as substrate (Km = 1.5 ��M; Vrel = 3 with Mg2+ as cofactor), the monoterpenes limonene (48%), terpinolene (15%), and myrcene (15%) are produced.

Photoassembly of the manganese cluster and oxygen evolution from monomeric and dimeric CP47 reaction center photosystem II complexes

Isolated subcomplexes of photosystem II from spinach (CP47RC), composed of D1, D2, cytochrome b559, CP47, and a number of hydrophobic small subunits but devoid of CP43 and the extrinsic proteins of the oxygen-evolving complex, were shown to reconstitute the Mn4Ca1Clx cluster of the water-splitting system and to evolve oxygen. The photoactivation process in CP47RC dimers proceeds by the same two-step mechanism as observed in PSII membranes and exhibits the same stoichiometry for Mn2+, but with a 10-fold lower affinity for Ca2+ and an increased susceptibility to photodamage. After the lower Ca2+ affinity and the 10-fold smaller absorption cross-section for photons in CP47 dimers is taken into account, the intrinsic rate constant for the rate-limiting calcium-dependent dark step is indistinguishable for the two systems. The monomeric form of CP47RC also showed capacity to photoactivate and catalyze water oxidation, but with lower activity than the dimeric form and increased susceptibility to photodamage. After optimization of the various parameters affecting the photoactivation process in dimeric CP47RC subcores, 18% of the complexes were functionally reconstituted and the quantum efficiency for oxygen production by reactivated centers approached 96% of that observed for reconstituted photosystem II-enriched membranes.

Bicarbonate is an essential constituent of the water-oxidizing complex of photosystem���II

It is shown that restoration of photoinduced electron flow and O2 evolution with Mn2+ in Mn-depleted photosystem II (PSII) membrane fragments isolated from spinach chloroplasts is considerably increased with bicarbonate in the region pH 5.0���8.0 in bicarbonate-depleted medium. In buffered solutions equilibrated with the atmosphere (nondepleted of bicarbonate), the bicarbonate effect is observed only at pH lower than the pK of H2CO3 dissociation (6.4), which indicates that HCO3��� is the essential species for the restoration effect. The addition of just 2 Mn2+ atoms per one PSII reaction center is enough for the maximal reactivation when bicarbonate is present in the medium. Analysis of bicarbonate concentration dependence of the restoration effect reveals two binding sites for bicarbonate with apparent dissociation constant (Kd) of ���2.5 ��M and 20���34 ��M when 2,6-dichloro-p-benzoquinone is used as electron acceptor, while in the presence of silicomolybdate only the latter one remains. Similar bicarbonate concentration dependence of O2 evolution was obtained in untreated Mn-containing PSII membrane fragments. It is suggested that the Kd of 20���34 ��M is associated with the donor side of PSII while the location of the lower Kd binding site is not quite clear. The conclusion is made that bicarbonate is an essential constituent of the water-oxidizing complex of PSII, important for its assembly and maintenance in the functionally active state.

The extended environment of mononuclear metal centers in protein���structures

The objectives of this and the following paper are to identify commonalities and disparities of the extended environment of mononuclear metal sites centering on Cu, Fe, Mn, and Zn. The extended environment of a metal site within a protein embodies at least three layers: the metal core, the ligand group, and the second shell, which is defined here to consist of all residues distant less than 3.5 ��� from some ligand of the metal core. The ligands and second-shell residues can be characterized in terms of polarity, hydrophobicity, secondary structures, solvent accessibility, hydrogen-bonding interactions, and membership in statistically significant residue clusters of different kinds. Findings include the following: (i) Both histidine ligands of type I copper ions exclusively attach the N��1 nitrogen of the histidine imidazole ring to the metal, whereas histidine ligands for all mononuclear iron ions and nearly all type II copper ions are ligated via the N��2 nitrogen. By contrast, multinuclear copper centers are coordinated predominantly by histidine N��2, whereas diiron histidine contacts are predominantly N��1. Explanations in terms of steric differences between N��1 and N��2 are considered. (ii) Except for blue copper (type I), the second-shell composition favors polar residues. (iii) For blue copper, the second shell generally contains multiple methionine residues, which are elements of a statistically significant histidine���cysteine���methionine cluster. Almost half of the second shell of blue copper consists of solvent-accessible residues, putatively facilitating electron transfer. (iv) Mononuclear copper atoms are never found with acidic carboxylate ligands, whereas single Mn2+ ion ligands are predominantly acidic and the second shell tends to be mostly buried. (v) The extended environment of mononuclear Fe sites often is associated with histidine���tyrosine or histidine���acidic clusters.

Explanation by the double-metal-ion mechanism of catalysis for the differential metal ion effects on the cleavage rates of 5���-oxy and 5���-thio substrates by a hammerhead���ribozyme

In a previous examination using natural all-RNA substrates that contained either a 5���-oxy or 5���-thio leaving group at the cleavage site, we demonstrated that (i) the attack by the 2���-oxygen at C17 on the phosphorus atom is the rate-limiting step only for the substrate that contains a 5���-thio group (R11S) and (ii) the departure of the 5��� leaving group is the rate-limiting step for the natural all-RNA substrate (R11O) in both nonenzymatic and hammerhead ribozyme-catalyzed reactions; the energy diagrams for these reactions were provided in our previous publication. In this report we found that the rate of cleavage of R11O by a hammerhead ribozyme was enhanced 14-fold when Mg2+ ions were replaced by Mn2+ ions, whereas the rate of cleavage of R11S was enhanced only 2.2-fold when Mg2+ ions were replaced by Mn2+ ions. This result appears to be exactly the opposite of that predicted from the direct coordination of the metal ion with the leaving 5���-oxygen, because a switch in metal ion specificity was not observed with the 5���-thio substrate. However, our quantitative analyses based on the previously provided energy diagram indicate that this result is in accord with the double-metal-ion mechanism of catalysis.

Interactions between Growth Factors and Integrins: Latent Forms of Transforming Growth Factor-�� Are Ligands for the Integrin ��v��1

The multipotential cytokine transforming growth factor-�� (TGF-��) is secreted in a latent form. Latency results from the noncovalent association of TGF-�� with its processed propeptide dimer, called the latency-associated peptide (LAP); the complex of the two proteins is termed the small latent complex. Disulfide bonding between LAP and latent TGF-�����binding protein (LTBP) produces the most common form of latent TGF-��, the large latent complex. The extracellular matrix (ECM) modulates the activity of TGF-��. LTBP and the LAP propeptides of TGF-�� (isoforms 1 and 3), like many ECM proteins, contain the common integrin-binding sequence RGD. To increase our understanding of latent TGF-�� function in the ECM, we determined whether latent TGF-��1 interacts with integrins. A549 cells adhered and spread on plastic coated with LAP, small latent complex, and large latent complex but not on LTBP-coated plastic. Adhesion was blocked by an RGD peptide, and cells were unable to attach to a mutant form of recombinant LAP lacking the RGD sequence. Adhesion was also blocked by mAbs to integrin subunits ��v and ��1. We purified LAP-binding integrins from extracts of A549 cells using LAP bound to Sepharose. ��v��1 eluted with EDTA. After purification in the presence of Mn2+, a small amount of ��v��5 was also detected. A549 cells migrated equally on fibronectin- and LAP-coated surfaces; migration on LAP was ��v��1 dependent. These results establish ��v��1 as a LAP-��1 receptor. Interactions between latent TGF-�� and ��v��1 may localize latent TGF-�� to the surface of specific cells and may allow the TGF-��1 gene product to initiate signals by both TGF-�� receptor and integrin pathways.

Modulation of In Vivo Migratory Function of ��2��1 Integrin in Mouse Liver

We report herein that expression of ��2��1 integrin increased human erythroleukemia K562 transfectant (KX2C2) cell movement after extravasation into liver parenchyma. In contrast, a previous study demonstrated that ��2��1 expression conferred a stationary phenotype to human rhabdomyosarcoma RD transfectant (RDX2C2) cells after extravasation into the liver. We therefore assessed the adhesive and migratory function of ��2��1 on KX2C2 and RDX2C2 cells using a ��2��1-specific stimulatory monoclonal antibody (mAb), JBS2, and a blocking mAb, BHA2.1. In comparison with RDX2C2 cells, KX2C2 were only weakly adherent to collagen and laminin. JBS2 stimulated ��2��1-mediated interaction of KX2C2 cells with both collagen and laminin resulting in increases in cell movement on both matrix proteins. In the presence of Mn2+, JBS2-stimulated adhesion on collagen beyond an optimal level for cell movement. In comparison, an increase in RDX2C2 cell movement on collagen required a reduction in its adhesive strength provided by the blocking mAb BHA2.1. Consistent with these in vitro findings, in vivo videomicroscopy revealed that ��2��1-mediated postextravasation cell movement of KX2C2 cells in the liver tissue could also be stimulated by JBS2. Thus, results demonstrate that ��2��1 expression can modulate postextravasation cell movement by conferring either a stationary or motile phenotype to different cell types. These findings may be related to the differing metastatic activities of different tumor cell types.

Modulatory Roles for Integrin Activation and the Synergy Site of Fibronectin during Matrix Assembly

Initiation of fibronectin (FN) matrix assembly is dependent on specific interactions between FN and cell surface integrin receptors. Here, we show that de novo FN matrix assembly exhibits a slow phase during initiation of fibrillogenesis followed by a more rapid growth phase. Mn2+, which acts by enhancing integrin function, increased the rate of FN fibril growth, but only after the initial lag phase. The RGD cell-binding sequence in type III repeat 10 is an absolute requirement for initiation by ��5��1 integrin. To investigate the role of the cell-binding synergy site in the adjacent repeat III9, a full-length recombinant FN containing a synergy mutation, FN(syn���), was tested for its ability to form fibrils. Mutation of this site drastically reduced FN assembly by CHO��5 cells. Only sparse short fibrils were formed even after prolonged incubation, indicating that FN(syn���) is defective in progression of the assembly process. These results show that the synergy site is essential for ��5��1-mediated accumulation of a FN matrix. However, the incorporation of FN(syn���) into fibrils and the deoxycholate-insoluble matrix could be stimulated by Mn2+. Therefore, exogenous activation of integrin receptors can overcome the requirement for FN���s synergy site as well as modulate the rate of FN matrix formation.

Comparison of the 5��� nuclease activities of Taq DNA polymerase and its isolated nuclease domain

Many eubacterial DNA polymerases are bifunctional molecules having both polymerization (P) and 5��� nuclease (N) activities, which are contained in separable domains. We previously showed that the DNA polymerase I of Thermus aquaticus (TaqNP) endonucleolytically cleaves DNA substrates, releasing unpaired 5��� arms of bifurcated duplexes. Here, we compare the substrate specificities of TaqNP and the isolated 5��� nuclease domain of this enzyme, TaqN. Both enzymes are significantly activated by primer oligonucleotides that are hybridized to the 3��� arm of the bifurcation; optimal stimulation requires overlap of the 3��� terminal nucleotide of the primer with the terminal base pair of the duplex, but the terminal nucleotide need not hybridize to the complementary strand in the substrate. In the presence of Mn2+ ions, TaqN can cleave both RNA and circular DNA at structural bifurcations. Certain anti-TaqNP mAbs block cleavage by one or both enzymes, whereas others can stimulate cleavage of nonoptimal substrates.