Substrate-induced Changes in Domain Interaction of Vacuolar H+-Pyrophosphatase

Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H⁺-pyrophosphatase (EC 3.6.1.1) from Vigna radiata. H⁺-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PP_i hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H⁺-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H⁺-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was ∼4 nm in length, ∼2 nm in width, and ∼1 nm in protrusion height. Upon extracting the H⁺-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H⁺-pyrophosphatase in the presence and absence of substrate and its analog.     

The Activity of the Root Vacuolar H+-Pyrophosphatase in Rye Plants Grown under Conditions Deficient in Mineral Nutrients

Compared to rye plants grown under normal conditions of mineralnutrients, those grown under deficient conditions of mineralnutrients were shown to have a high potential activity of theroot vacuolar H⁺-pyrophos-phatase (H⁺-PPiase), with a low levelof PP₁ in roots. Im-munoblot analysis suggested a qualitativechange of the enzyme. (Received April 3, 1998; )     

Accumulation of Vacuolar H+-Pyrophosphatase and H+-ATPase during Reformation of the Central Vacuole in Germinating Pumpkin Seeds

Protein storage vacuoles were examined for the induction of H+-pyrophosphatase (H+-PPase), H+-ATPase, and a membrane integral protein of 23 kD after seed germination. Membranes of protein storage vacuoles were prepared from dry seeds and etiolated cotyledons of pumpkin (Cucurbita sp.). Membrane vesicles from etiolated cotyledons had ATP- and pyrophosphate-dependent H+-transport activities. H+-ATPase activity was sensitive to nitrate and bafilomycin, and H+-PPase activity was stimulated by potassium ion and inhibited by dicyclohexylcarbodiimide. The activities of both enzymes increased after seed germination. On immunoblot analysis, the 73-kD polypeptide of H+-PPase and the two major subunits, 68 and 57 kD, of vacuolar H+-ATPase were detected in the vacuolar membranes of cotyledons, and the levels of the subunits of enzymes increased parallel to those of enzyme activities. Small amounts of the subunits of the enzymes were detected in dry cotyledons. Immunocytochemical analysis of the cotyledonous cells with anti-H+-PPase showed the close association of H+-PPase to the membranes of protein storage vacuoles. In endosperms of castor bean (Ricinus communis), both enzymes and their subunits increased after germination. Furthermore, the vacuolar membranes from etiolated cotyledons of pumpkin had a polypeptide that cross-reacted with antibody against a 23-kD membrane protein of radish vacuole, VM23, but the membranes of dry cotyledons did not. The results from this study suggest that H+-ATPase, H+-PPase, and VM23 are expressed and accumulated in the membranes of protein storage vacuoles after seed germination. Overall, the findings indicate that the membranes of protein storage vacuoles are transformed into those of central vacuoles during the growth of seedlings.     

Dynamics of Vacuoles and H+-Pyrophosphatase Visualized by Monomeric Green Fluorescent Protein in Arabidopsis: Artifactual Bulbs and Native Intravacuolar Spherical Structures

We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H⁺-pyrophosphatase (H+-PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H+-PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H+-PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H⁺-PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H⁺-PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.     

Structural Studies of the Vacuolar H+-Pyrophosphatase: Sequence Analysis and Identification of the Residues Modified by Fluorescent Cyclohexylcarbodiimide and Maleimide

We determined the amino acid residues of the H⁺-translocatinginorganic pyrophosphatase (H⁺-PPase) of pumpkin which are covalentlylabeled by two fluorescent labeling reagents; N-cyclohexyl-.N'-[4-(dimethylamino)-     

Changes in H+-Pumps and a Tonoplast Intrinsic Protein of Vacuolar Membranes during the Development of Pear Fruit

Vacuolar H⁺-ATPase (V-ATPase) was purified from pear fruit andantibodies were raised against the subunits of 55 and 33 kDa.Antibodies against mung bean H⁺-pyro-phosphatase (V-PPase) andradish VM23, which is a tonoplast intrinsic protein (TIP) anda water channel, cross-reacted with the vacuolar membrane proteinsof pear fruit. To clarify the roles of these proteins in developmentof pear fruit, we determined their levels relative to the totalamount of protein by immunoblot analysis. The levels of subunitsof the V-ATPase increased with fruit development. By contrast,the level of V-PPase was particularly high at the cell-divisionstage and remained almost the same at other stages. The changesin the activities of V-ATPase and V-PPase corresponded to thosein their protein levels. The ratio of V-PPase activity to V-ATPaseactivity indicated that V-PPase is a major H⁺-pump of the vacuolarmembranes of young fruit and that the contribution of V-ATPaseincreases with fruit development, finally, V-ATPase becomesthe major H⁺-pump during the later stages of fruit development.The level of a protein analogous to VM23 (VM23P) was especiallyhigh during the active cell-expansion stage in young fruit,and VM23P might, therefore, play an important role in the rapidexpansion of cells as a vacuolar water channel. Our resultsshow that the levels of V-ATPase, V-PPase and VM23P change differentlyand reflect the roles of the respective proteins in the developmentof pear fruit. ³Research Fellow of the Japan Society for the Promotion of Science ⁴Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan     

Characteristics of Root Plasma Membrane ATPase and H+- extrusion in a K+- deficit Tolerant Rice Variety

The characteristics of root plasma membrane ATPase (PM-ATPase) of "Weiyou 49", a K+ -deficit tolerant rice (Oryza sativa L. ) variety and of "Yuanyou 1", a K+ -deficit non-tolerant rice variety, had some similarities:Their optimum pH value were both about 6.0; Their activities reached the maximum at ATP concentration of 3 mmol/L; Km was 0.85 mmol/L and external K+ stimulated their activities. However, when [K+ ] was less than or equal to 50 mmol/L in the medium, the increasing of K + stimulated the activity of the PM-ATPase of "Weiyou 49" much more than that of "Yuanyou 1". When [K+ ] was between 100 to 200 mmol/L, the difference of the PM-AT- Pase activities decreased between the two rice varieties caused by K + stimulation. The basic H + extrusion of the two varieties had no apparent difference, but the H + extrusion stimulated by K + was different. The H+ extrusion of "Weiyou 49" was relatively more sensitive to external K+ . The experiment using inhibitors showed that there were close relationship between the PM-ATPase activi- ties stimulated by K+ and K+ uptake in the two varieties. The inhibition of PM-ATPase activity and H+ -extrusion stimulated by K+ reduced the K+ uptake of the root segments in both varieties. So the possible reason for "Weiyou 49" growing well in the low external K+ was that its PM-ATPase and H+ extrusion was more sensitive to external K+ , especially when [K+ ] was low.     

Reversibility of H+-ATPase and H+-Pyrophosphatase in Tonoplast Vesicles from Maize Coleoptiles and Seeds

Tonoplast-enriched vesicles isolated from maize (Zea mays L.) coleoptiles and seeds synthesize ATP from ADP and inorganic phosphate (Pi) and inorganic pyrophosphate from Pi. The synthesis is consistent with reversal of the catalytic cycle of the H⁺-ATPase and H⁺-pyrophosphatase (PPase) vacuolar membrane-bound enzymes. This was monitored by measuring the exchange reaction that leads to ³²Pi incorporation into ATP or inorganic pyrophosphate. The reversal reactions of these enzymes were dependent on the proton gradient formed across the vesicle membrane and were susceptible to the uncoupler carbonyl cyanide p(trifluoromethoxy)-phenylhydrazone and the detergent Triton X-100. Comparison of the two H⁺ pumps showed that the H⁺-ATPase was more active than H⁺-PPase in coleoptile tonoplast vesicles, whereas in seed vesicles H⁺-PPase activity was clearly dominant. These findings may reflect the physiological significance of these enzymes in different tissues at different stages of development and/or differentiation.     

液泡膜H+-ATPase在植物的非生物胁迫响应和信号转导中的作用

液泡膜H^＋-ATPase是一种多亚基复合体,在植物受到非生物胁迫后,其对逆境信号的感知转导即做出相应的变化。在Ca^2＋通道、ABA信号通路及盐过敏感途径等信号传递的过程中,都有V-ATPase的参与。文章将对这一领域的研究进展进行介绍。     

Assembly and Regulation of the Yeast Vacuolar H+-ATPase

The yeast vacuolar proton-translocating ATPase (V-ATPase) is an excellent model for V-ATPases in all eukaryotic cells. Activity of the yeast V-ATPase is reversibly down-regulated by disassembly of the peripheral (V₁) sector, which contains the ATP-binding sites, from the membrane (V₀) sector, which contains the proton pore. A similar regulatory mechanism has been found in Manduca sexta and is believed to operate in other eukaryotes. We are interested in the mechanism of reversible disassembly and its implications for V-ATPase structure. In this review, we focus on (1) characterization of the yeast V-ATPase stalk subunits, which form the interface between V₁ and V₀, (2) potential mechanisms of silencing ATP hydrolytic activity in disassembled V₁ sectors, and (3) the structure and function of RAVE, a recently discovered complex that regulates V-ATPase assembly.     

Biosynthesis and Regulation of the Yeast Vacuolar H+-ATPase

The yeast V-ATPase is highly similar to V-ATPases of higher organismsand has proved to be a biochemically and genetically accessible model formany aspects of V-ATPase function. Like other V-ATPases, the yeast enzymeconsists of a complex of peripheral membrane proteins, the V₁sector, attached to a complex of integral membrane subunits, theV₀ sector. Multiple pathways for biosynthetic assembly of theenzyme appear to be available to cells containing a full complement ofsubunits and enzyme activity may be further controlled during biosynthesis bya protease activity localized to the late Golgi apparatus. Surprisingly, theassembled V-ATPase is not a static structure. Instead, fully assembledV₁V₀ complexes appear to exist in a dynamic equilibriumwith inactive cytosolic V₁ and membrane-bound V₀complexes and this equilibrium can be rapidly shifted in response to changesin carbon source. The reversible disassembly of the yeast V-ATPase may be anovel regulatory mechanism, common to V-ATPases, that works in vivoin coordination with many other regulatory mechanisms.     

Vacuolar Cation/H+ Antiporters of Saccharomyces cerevisiae

We previously demonstrated that Saccharomyces cerevisiae vnx1Δ mutant strains displayed an almost total loss of Na⁺ and K⁺/H⁺ antiporter activity in a vacuole-enriched fraction. However, using different in vitro transport conditions, we were able to reveal additional K⁺/H⁺ antiporter activity. By disrupting genes encoding transporters potentially involved in the vnx1 mutant strain, we determined that Vcx1p is responsible for this activity. This result was further confirmed by complementation of the vnx1Δvcx1Δ nhx1Δ triple mutant with Vcx1p and its inactivated mutant Vcx1p-H303A. Like the Ca²⁺/H⁺ antiporter activity catalyzed by Vcx1p, the K⁺/H⁺ antiporter activity was strongly inhibited by Cd²⁺ and to a lesser extend by Zn²⁺. Unlike as previously observed for NHX1 or VNX1, VCX1 overexpression only marginally improved the growth of yeast strain AXT3 in the presence of high concentrations of K⁺ and had no effect on hygromycin sensitivity. Subcellular localization showed that Vcx1p and Vnx1p are targeted to the vacuolar membrane, whereas Nhx1p is targeted to prevacuoles. The relative importance of Nhx1p, Vnx1p, and Vcx1p in the vacuolar accumulation of monovalent cations will be discussed.     

Engineering a Novel Multifunctional Green Fluorescent Protein Tag for a Wide Variety of Protein Research

Background

Genetically encoded tag is a powerful tool for protein research. Various kinds of tags have been developed: fluorescent proteins for live-cell imaging, affinity tags for protein isolation, and epitope tags for immunological detections. One of the major problems concerning the protein tagging is that many constructs with different tags have to be made for different applications, which is time- and resource-consuming.

Methodology/Principal Findings

Here we report a novel multifunctional green fluorescent protein (mfGFP) tag which was engineered by inserting multiple peptide tags, i.e., octa-histidine (8×His), streptavidin-binding peptide (SBP), and c-Myc tag, in tandem into a loop of GFP. When fused to various proteins, mfGFP monitored their localization in living cells. Streptavidin agarose column chromatography with the SBP tag successfully isolated the protein complexes in a native form with a high purity. Tandem affinity purification (TAP) with 8×His and SBP tags in mfGFP further purified the protein complexes. mfGFP was clearly detected by c-Myc-specific antibody both in immunofluorescence and immuno-electron microscopy (EM). These findings indicate that mfGFP works well as a multifunctional tag in mammalian cells. The tag insertion was also successful in other fluorescent protein, mCherry.

Conclusions and Significance

The multifunctional fluorescent protein tag is a useful tool for a wide variety of protein research, and may have the advantage over other multiple tag systems in its higher expandability and compatibility with existing and future tag technologies.     

Vegetative and Seed-Specific Forms of Tonoplast Intrinsic Protein in the Vacuolar Membrane of Arabidopsis thaliana

Reports from a number of laboratories describe the presence of a family of proteins (the major intrinsic protein family) in a variety of organisms. These proteins are postulated to form channels that function in metabolite transport. In plants, this family is represented by the product of NOD26, a nodulation gene in soybean that encodes a protein of the peribacteroid membrane, and tonoplast intrinsic protein (TIP), an abundant protein in the tonoplast of protein storage vacuoles of bean seeds (KD Johnson, H Höfte, MJ Chrispeels [1990] Plant Cell 2: 525-532). Other homologs that are induced by water stress in pea and in Arabidopsis thaliana and that are expressed in the roots of tobacco have been reported, but the location of the proteins they encode is not known. We now report the presence and derived amino acid sequences of two different TIP proteins in A. thaliana. α-TIP is a seed-specific protein that has 68% amino acid sequence identity with bean seed TIP; γ-TIP is expressed in the entire vegetative body of A. thaliana and has 58% amino acid identity with bean seed TIP. Both proteins are associated with the tonoplast. Comparisons of the derived amino acid sequences of the seven known plant proteins in the major intrinsic protein family show that genes with similar expression patterns (e.g. water stress-induced or seed specific) are more closely related to each other than the three A. thaliana homologs are related. We propose that the nonoverlapping gene expression patterns reported here, and the evolutionary relationships indicated by the phylogenetic tree, suggest a functional specialization of these proteins.     

A Vacuolar-Type H+-ATPase in a Nonvacuolar Organelle Is Required for the Sorting of Soluble Vacuolar Protein Precursors in Tobacco Cells

In plant cells, vacuolar matrix proteins are separated from the secretory proteins at the Golgi complex for transport to the vacuoles. To investigate the involvement of vacuolar-type ATPase (V-ATPase) in the vacuolar targeting of soluble proteins, we analyzed the effects of bafilomycin A1 and concanamycin A on the transport of vacuolar protein precursors in tobacco cells. Low concentrations of these inhibitors caused the missorting of several vacuolar protein precursors; sorting was more sensitive to concanamycin A than to bafilomycin A1. Secretion of soluble proteins from tobacco cells was also inhibited by bafilomycin A1 and concanamycin A. We next analyzed the subcellular localization of V-ATPase. V-ATPase was found in a wide variety of endomembrane organelles. Both ATPase activity and ATP-dependent proton-pumping activity in the Golgi-enriched fraction were more sensitive to concanamycin A than to bafilomycin A1, whereas these activities in the tonoplast fraction were almost equally sensitive to both reagents. Our observations indicate that the V-ATPase in the organelle that was recovered in the Golgi-enriched fraction is required for the transport of vacuolar protein precursors and that this V-ATPase is distinguishable from the tonoplast-associated V-ATPase.     

A Pumpkin 72-kDa Membrane Protein of Precursor-Accumulating Vesicles Has Characteristics of a Vacuolar Sorting Receptor

Precursor-accumulating (PAC) vesicles were previously shownto mediate the transport of the precursor of a major storageprotein (pro2S albumin) to protein-storage vacu-oles in developingpumpkin cotyledons. In this study, we characterized two homologousproteins from PAC vesicles, a 72 kDa protein (PV72) and an 82kDa protein (PV82). PV72 and PV82 showed an ability to bindto peptides derived from both an internal propeptide and a C-terminalpeptide of pro2S albumin. PV72 was predicted to be a type Iintegral membrane protein with epidermal growth factor (EGF)-likemotifs. These results suggest that PV72 and PV82 are potentialsorting receptors for 2S albumin to protein-storage vacuoles. (Received August 25, 1997; Accepted October 17, 1997)     

Isolation and Functional Validation of Salinity and Osmotic Stress Inducible Promoter from the Maize Type-II H+-Pyrophosphatase Gene by Deletion Analysis in Transgenic Tobacco Plants

Salinity and drought severely affect both plant growth and productivity, making the isolation and characterization of salinity- or drought-inducible promoters suitable for genetic improvement of crop resistance highly desirable. In this study, a 1468-bp sequence upstream of the translation initiation codon ATG of the promoter for ZmGAPP (maize Type-II H⁺-pyrophosphatase gene) was cloned. Nine 5´ deletion fragments (D1–D9) of different lengths of the ZmGAPP promoter were fused with the GUS reporter and translocated into tobacco. The deletion analysis showed that fragments D1–D8 responded well to NaCl and PEG stresses, whereas fragment D9 and CaMV 35S did not. The D8 segment (219 bp; -219 to -1 bp) exhibited the highest promoter activity of all tissues, with the exception of petals among the D1–D9 transgenic tobacco, which corresponds to about 10% and 25% of CaMV 35S under normal and NaCl or PEG stress conditions, respectively. As such, the D8 segment may confer strong gene expression in a salinity and osmotic stress inducible manner. A 71-bp segment (-219 to -148 bp) was considered as the key region regulating ZmGAPP response to NaCl or PEG stress, as transient transformation assays demonstrated that the 71-bp sequence was sufficient for the salinity or osmotic stress response. These results enhance our understanding of the molecular mechanisms regulating ZmGAPP expression, and that the D8 promoter would be an ideal candidate for moderating expression of drought and salinity response genes in transgenic plants.