GTPase Activity and Biochemical Characterization of a Recombinant Cotton Fiber Annexin1

A cDNA encoding annexin was isolated from a cotton (Gossypium hirsutum) fiber cDNA library. The cDNA was expressed in Escherichia coli, and the resultant recombinant protein was purified. We then investigated some biochemical properties of the recombinant annexin based on the current understanding of plant annexins. An “add-back experiment” was performed to study the effect of the recombinant annexin on β-glucan synthase activity, but no effect was found. However, it was found that the recombinant annexin could display ATPase/GTPase activities. The recombinant annexin showed much higher GTPase than ATPase activity. Mg2+ was essential for these activities, whereas a high concentration of Ca2+ was inhibitory. A photolabeling assay showed that this annexin could bind GTP more specifically than ATP. The GTP-binding site on the annexin was mapped into the carboxyl-terminal fourth repeat of annexin from the photolabeling experiment using domain-deletion mutants of this annexin. Northern-blot analysis showed that the annexin gene was highly expressed in the elongation stages of cotton fiber differentiation, suggesting a role of this annexin in cell elongation.

The Two Major Types of Plant Plasma Membrane H+-ATPases Show Different Enzymatic Properties and Confer Differential pH Sensitivity of Yeast Growth1

The proton-pumping ATPase (H+-ATPase) of the plant plasma membrane is encoded by two major gene subfamilies. To characterize individual H+-ATPases, PMA2, an H+-ATPase isoform of tobacco (Nicotiana plumbaginifolia), was expressed in Saccharomyces cerevisiae and found to functionally replace the yeast H+-ATPase if the external pH was kept above 5.0 (A. de Kerchove d'Exaerde, P. Supply, J.P. Dufour, P. Bogaerts, D. Thinès, A. Goffeau, M. Boutry [1995] J Biol Chem 270: 23828–23837). In the present study we replaced the yeast H+-ATPase with PMA4, an H+-ATPase isoform from the second subfamily. Yeast expressing PMA4 grew at a pH as low as 4.0. This was correlated with a higher acidification of the external medium and an approximately 50% increase of ATPase activity compared with PMA2. Although both PMA2 and PMA4 had a similar pH optimum (6.6–6.8), the profile was different on the alkaline side. At pH 7.2 PMA2 kept more than 80% of the maximal activity, whereas that of PMA4 decreased to less than 40%. Both enzymes were stimulated up to 3-fold by 100 μg/mL lysophosphatidylcholine, but this stimulation vanished at a higher concentration in PMA4. These data demonstrate functional differences between two plant H+-ATPases expressed in the same heterologous host. Characterization of two PMA4 mutants selected to allow yeast growth at pH 3.0 revealed that mutations within the carboxy-terminal region of PMA4 could still improve the enzyme, resulting in better growth of yeast cells.

Inhibitory Regulation of Higher-Plant Myosin by Ca2+ Ions1

Myosin isolated from the pollen tubes of lily (Lilium longiflorum) is composed of a 170-kD heavy chain (E. Yokota and T. Shimmen [1994] Protoplasma 177: 153–162). Both the motile activity in vitro and the F-actin-stimulated ATPase activity of this myosin were inhibited by Ca2+ at concentrations higher than 10−6 m. In the Ca2+ range between 10−6 and 10−5 m, inhibition of the motile activity was reversible. In contrast, inhibition by more than 10−5 m Ca2+ was not reversible upon Ca2+ removal. An 18-kD polypeptide that showed the same mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as that of spinach calmodulin (CaM) was present in this myosin fraction. This polypeptide showed a mobility shift in sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a Ca2+-dependent manner. Furthermore, this polypeptide was recognized by antiserum against spinach CaM. By immunoprecipitation using antiserum against the 170-kD heavy chain, the 18-kD polypeptide was coprecipitated with the 170-kD heavy chain, provided that the Ca2+ concentration was low, indicating that this 18-kD polypeptide is bound to the 170-kD myosin heavy chain. However, the 18-kD polypeptide was dissociated from the 170-kD heavy chain at high Ca2+ concentrations, which irreversibly inhibited the motile activity of this myosin. From these results, it is suggested that the 18-kD polypeptide, which is likely to be CaM, is associated with the 170-kD heavy chain as a light chain. It is also suggested that this polypeptide is involved in the regulation of this myosin by Ca2+. This is the first biochemical basis, to our knowledge, for Ca2+ regulation of cytoplasmic streaming in higher plants.

Visualization of unwinding activity of duplex RNA by DbpA, a DEAD box helicase, at single-molecule resolution by atomic force microscopy

The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized. Herein, we show by using biochemical assays and atomic force microscopy that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. This work presents an attempt to investigate the action of DEAD box proteins by a single-molecule visualization methodology. Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA. Specifically, we could differentiate between the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases.

The HMG-domain protein BAP111 is important for the function of the BRM chromatin-remodeling complex in vivo

The Drosophila trithorax group gene brahma (brm) encodes the ATPase subunit of a SWI/SNF-like chromatin-remodeling complex. A key question about chromatin-remodeling complexes is how they interact with DNA, particularly in the large genomes of higher eukaryotes. Here, we report the characterization of BAP111, a BRM-associated protein that contains a high mobility group (HMG) domain predicted to bind distorted or bent DNA. The presence of an HMG domain in BAP111 suggests that it may modulate interactions between the BRM complex and chromatin. BAP111 is an abundant nuclear protein that is present in all cells throughout development. By using gel filtration chromatography and immunoprecipitation assays, we found that the majority of BAP111 protein in embryos is associated with the BRM complex. Furthermore, heterozygosity for BAP111 enhanced the phenotypes resulting from a partial loss of brm function. These data demonstrate that the BAP111 subunit is important for BRM complex function in vivo.

Large conformational changes of the ɛ subunit in the bacterial F1F0 ATP synthase provide a ratchet action to regulate this rotary motor enzyme

The F1F0 ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the γ and ɛ subunits in the F1 part and c subunit ring in the F0 part, rotates relative to a stator composed of α3β3δab2 during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the ɛ subunit plays a key role by acting as a switch of this motor. Two different arrangements of the ɛ subunit have been visualized recently. The first has been observed in beef heart mitochondrial F1-ATPase where the C-terminal portion is arranged as a two-α-helix hairpin structure that extends away from the α3β3 region, and toward the position of the c subunit ring in the intact F1F0. The second arrangement was observed in a structure determination of a complex of the γ and ɛ subunits of the Escherichia coli F1-ATPase. In this, the two C-terminal helices are apart and extend along the γ to interact with the α and β subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F1F0, confirming that both conformations of the ɛ subunit exist in the enzyme complex. With the C-terminal domain of ɛ toward the F0, ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F1 part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the ɛ subunit in the F1F0 complex and argue for a ratchet function of this subunit.

Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the α- and β-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Identification and characterization of a lysosomal transporter for small neutral amino acids

In eukaryotic cells, lysosomes represent a major site for macromolecule degradation. Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters. Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases. However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level. In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters. LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons. Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as γ-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport. Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker. LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles. Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family.

Afipia felis induces uptake by macrophages directly into a nonendocytic compartment

Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease. A. felis can survive and multiply in several mammalian cell types, including macrophages, but the precise intracellular compartmentalization of A. felis-containing phagosomes is unknown. Here, we demonstrate that, in murine macrophages, most A. felis-containing phagosomes exclude lysosomal tracer loaded into macrophage lysosomes before, as well as endocytic tracer loaded after, establishment of an infection. Established Afipia-containing phagosomes possess neither early endosomal marker proteins [early endosome antigen 1 (EEA1), Rab5, transferrin receptor, trytophane aspartate containing coat protein (TACO)] nor late endosomal or lysosomal proteins [cathepsin D, β-glucuronidase, vacuolar proton-pumping ATPase, rab7, mannose-6-phosphate receptor, vesicle-associated membrane protein 8, lysosome-associated membrane proteins LAMP-1 and LAMP-2]. Those bacteria that will be found in a nonendosomal compartment enter the macrophage via an EEA1-negative compartment, which remains negative for LAMP-1. The smaller subpopulation of afipiae whose phagosomes will be part of the endocytic system enters into an EEA1-positive compartment, which also subsequently acquires LAMP-1. Killing of Afipia or opsonization with immune antibodies leads to a strong increase in the percentage of A. felis-containing phagosomes that interact with the endocytic system. We conclude that most phagosomes containing A. felis are disconnected from the endosome–lysosome continuum, that their unusual compartmentalization is decided at uptake, and that this compartmentalization requires bacterial viability.

A Chloroplast DNA Helicase II from Pea That Prefers Fork-Like Replication Structures

A DNA helicase, called chloroplast DNA (ctDNA) helicase II, was purified to apparent homogeneity from pea (Pisum sativum). The enzyme contained intrinsic, single-stranded, DNA-dependent ATPase activity and an apparent molecular mass of 78 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DNA helicase was markedly stimulated by DNA substrates with fork-like replication structures. A 5′-tailed fork was more active than the 3′-tailed fork, which itself was more active than substrates without a fork. The direction of unwinding was 3′ to 5′ along the bound strand, and it failed to unwind blunt-ended duplex DNA. DNA helicase activity required only ATP or dATP hydrolysis. The enzyme also required a divalent cation (Mg2+>Mn2+>Ca2+) for its unwinding activity and was inhibited at 200 mm KCl or NaCl. This enzyme could be involved in the replication of ctDNA. The DNA major groove-intercalating ligands nogalamycin and daunorubicin were inhibitory to unwinding (Ki approximately 0.85 μm and 2.2 μm, respectively) and ATPase (Ki approximately 1.3 μm and 3.0 μm, respectively) activities of pea ctDNA helicase II, whereas ellipticine, etoposide (VP-16), and camptothecin had no effect on the enzyme activity. These ligands may be useful in further studies of the mechanisms of chloroplast helicase activities.

Heterogeneity of Mitochondrial Protein Biogenesis during Primary Leaf Development in Barley1

The natural developmental gradient of light-grown primary leaves of barley (Hordeum vulgare L.) was used to analyze the biogenesis of mitochondrial proteins in relation to the age and physiological changes within the leaf. The data indicate that the protein composition of mitochondria changes markedly during leaf development. Three distinct patterns of protein development were noted: group A proteins, consisting of the E1 β-subunit of the pyruvate dehydrogenase complex, ORF156, ORF577, alternative oxidase, RPS12, cytochrome oxidase subunits II and III, malic enzyme, and the α- and β-subunits of F1-ATPase; group B proteins, consisting of the E1 α-subunit of the pyruvate dehydrogenase complex, isocitrate dehydrogenase, HSP70A, cpn60C, and cpn60B; and group C proteins, consisting of the four subunits of the glycine decarboxylase complex (P, H, T, and L proteins), fumarase, and formate dehydrogenase. All of the proteins increased in concentration from the basal meristem to the end of the elongation zone (20.0 mm from the leaf base), whereupon group A proteins decreased, group B proteins increased to a maximum at 50 mm from the leaf base, and group C proteins increased to a maximum at the leaf tip. This study provides evidence of a marked heterogeneity of mitochondrial protein composition, reflecting a changing function as leaf cells develop photosynthetic and photorespiratory capacity.

A High-Affinity Ca2+ Pump, ECA1, from the Endoplasmic Reticulum Is Inhibited by Cyclopiazonic Acid but Not by Thapsigargin1

To identify and characterize individual Ca2+ pumps, we have expressed an Arabidopsis ECA1 gene encoding an endoplasmic reticulum-type Ca2+-ATPase homolog in the yeast (Saccharomyces cerevisiae) mutant K616. The mutant (pmc1pmr1cnb1) lacks a Golgi and a vacuolar membrane Ca2+ pump and grows very poorly on Ca2+-depleted medium. Membranes isolated from the mutant showed high H+/Ca2+-antiport but no Ca2+-pump activity. Expression of ECA1 in endomembranes increased mutant growth by 10- to 20-fold in Ca2+-depleted medium. 45Ca2+ pumping into vesicles from ECA1 transformants was detected after the H+/Ca2+-antiport activity was eliminated with bafilomycin A1 and gramicidin D. The pump had a high affinity for Ca2+ (Km = 30 nm) and displayed two affinities for ATP (Km of 20 and 235 μm). Cyclopiazonic acid, a specific blocker of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, inhibited Ca2+ transport (50% inhibition dose = 3 nmol/mg protein), but thapsigargin (3 μm) did not. Transport was insensitive to calmodulin. These results suggest that this endoplasmic reticulum-type Ca2+-ATPase could support cell growth in plants as in yeast by maintaining submicromolar levels of cytosolic Ca2+ and replenishing Ca2+ in endomembrane compartments. This study demonstrates that the yeast K616 mutant provides a powerful expression system to study the structure/function relationships of Ca2+ pumps from eukaryotes.

Evidence That Auxin-Induced Growth of Tobacco Leaf Tissues Does Not Involve Cell Wall Acidification1

Interveinal strips (10 × 1.5 mm) excised from growing tobacco (Nicotiana tabacum L. cv Xanthi) leaves have an auxin-specific, epinastic growth response that is developmentally regulated and is not the result of ethylene induction (C.P. Keller, E. Van Volkenburgh [1997] Plant Physiol 113: 603–610). We report here that auxin (10 μm naphthalene acetic acid) treatment of strips does not result in plasma membrane hyperpolarization or detectable proton efflux. This result is in contrast to the expected responses elicited by 1 μm fusicoccin (FC) treatment, which in other systems mimics auxin growth promotion through stimulation of the plasma membrane H+-ATPase and resultant acid wall loosening; FC produced both hyperpolarization and proton efflux in leaf strips. FC-induced growth was much more inhibited by a strong neutral buffer than was auxin-induced growth. Measurements of the osmotic concentration of strips suggested that osmotic adjustment plays no role in the auxin-induced growth response. Although cell wall loosening of some form appears to be involved, taken together, our results suggest that auxin-induced growth stimulation of tobacco leaf strips results primarily from a mechanism not involving acid growth.

Initial Steps in the Assembly of the Vacuole-Type H+-ATPase1

The plant vacuole is acidified by a complex multimeric enzyme, the vacuole-type H+-ATPase (V-ATPase). The initial association of ATPase subunits on membranes was studied using an in vitro assembly assay. The V-ATPase assembled onto microsomes when V-ATPase subunits were supplied. However, when the A or B subunit or the proteolipid were supplied individually, only the proteolipid associated with membranes. By using poly(A+) RNA depleted in the B subunit and proteolipid subunit mRNA, we demonstrated A subunit association with membranes at substoichiometric amounts of the B subunit or the 16-kD proteolipid. These data suggest that poly(A+) RNA-encoded proteins are required to catalyze the A subunit membrane assembly. Initial events were further studied by in vivo protein labeling. Consistent with a temporal ordering of V-ATPase assembly, membranes contained only the A subunit at early times; at later times both the A and B subunits were found on the membranes. A large-mass ATPase complex was not efficiently formed in the absence of membranes. Together, these data support a model whereby the A subunit is first assembled onto the membrane, followed by the B subunit.

Characterization of a Red Beet Protein Homologous to the Essential 36-Kilodalton Subunit of the Yeast V-Type ATPase1

V-type proton-translocating ATPases (V-ATPases) (EC 3.6.1.3) are electrogenic proton pumps involved in acidification of endomembrane compartments in all eukaryotic cells. V-ATPases from various species consist of 8 to 12 polypeptide subunits arranged into an integral membrane proton pore sector (V0) and a peripherally associated catalytic sector (V1). Several V-ATPase subunits are functionally and structurally conserved among all species examined. In yeast, a 36-kD peripheral subunit encoded by the yeast (Saccharomyces cerevisiae) VMA6 gene (Vma6p) is required for stable assembly of the V0 sector as well as for V1 attachment. Vma6p has been characterized as a nonintegrally associated V0 subunit. A high degree of sequence similarity among Vma6p homologs from animal and fungal species suggests that this subunit has a conserved role in V-ATPase function. We have characterized a novel Vma6p homolog from red beet (Beta vulgaris) tonoplast membranes. A 44-kD polypeptide cofractionated with V-ATPase upon gel-filtration chromatography of detergent-solubilized tonoplast membranes and was specifically cross-reactive with anti-Vma6p polyclonal antibodies. The 44-kD polypeptide was dissociated from isolated tonoplast preparations by mild chaotropic agents and thus appeared to be nonintegrally associated with the membrane. The putative 44-kD homolog appears to be structurally similar to yeast Vma6p and occupies a similar position within the holoenzyme complex.