Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus

Background information. The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe ( 2004 ) Cell Struct. Funct. 29 , 85–90]. Results. In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295‐amino‐acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn‐Pro‐Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti‐ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V‐ATPase (vacuolar H⁺‐ATPase) on the vesicle membrane around the CV was also detected. Conclusions. Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V‐ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.     

Tissue Distribution, Effects of Salinity Acclimation, and Ontogeny of Aquaporin 3 in the Marine Teleost, Silver Sea Bream (Sparus sarba)

The purpose of the present study was to ascertain the tissue-specific expression of the water channel protein, aquaporin 3 (AQP3), during salinity acclimation and larval development of silver sea bream (Sparus sarba). A cDNA fragment encoding aquaporin 3 (aqp3) from silver sea bream gill was cloned and from the deduced amino acid sequence a polyclonal antibody was prepared. AQP3 was found to be present in gill, kidney, liver, brain, heart, and spleen but not in whole blood. The abundance of AQP3 was significantly highest in gills of hypoosmotic (6 ppt) and isoosmotic (12 ppt) acclimated sea bream when compared to seawater (33 ppt) and hypersaline (50 ppt)- acclimated sea bream. Spleen tissue also displayed significantly high levels of AQP3 protein in hypoosmotic and isoosmotic salinities whereas the AQP3 abundance in brain, liver, heart, and kidney remained unchanged across the range of salinities tested. The ontogenetic profile of AQP3 was also investigated from developing sea bream larvae and AQP3 was first detected at 14 days posthatch (dph) and increased steadily up to 28–46 dph. In conclusion, this study has demonstrated that AQP3 expression is modulated in gill and spleen tissue of salinity acclimated sea bream and that it can be detected relatively early during larval development.     

Molecular Characterization of Branchial aquaporin 1aa and Effects of Seawater Acclimation,Emersion or Ammonia Exposure on Its mRNA Expression in the Gills,Gut, Kidney and Skin of the Freshwater Climbing Perch,Anabas testudineus

We obtained a full cDNA coding sequence of aquaporin 1aa (aqp1aa) from the gills of the freshwater climbing perch, Anabas testudineus, which had the highest expression in the gills and skin, suggesting an important role of Aqp1aa in these organs. Since seawater acclimation had no significant effects on the branchial and intestinal aqp1aa mRNA expression, and since the mRNA expression of aqp1aa in the gut was extremely low, it can be deduced that Aqp1aa, despite being a water channel, did not play a significant osmoregulatory role in A. testudineus. However, terrestrial exposure led to significant increases in the mRNA expression of aqp1aa in the gills and skin of A. testudineus. Since terrestrial exposure would lead to evaporative water loss, these results further support the proposition that Aqp1aa did not function predominantly for the permeation of water through the gills and skin. Rather, increased aqp1aa mRNA expression might be necessary to facilitate increased ammonia excretion during emersion, because A. testudineus is known to utilize amino acids as energy sources for locomotor activity with increased ammonia production on land. Furthermore, ammonia exposure resulted in significant decreases in mRNA expression of aqp1aa in the gills and skin of A. testudineus, presumably to reduce ammonia influx during ammonia loading. This corroborates previous reports on AQP1 being able to facilitate ammonia permeation. However, a molecular characterization of Aqp1aa from A. testudineus revealed that its intrinsic aquapore might not facilitate NH₃ transport. Hence, ammonia probably permeated the central fifth pore of the Aqp1aa tetramer as suggested previously. Taken together, our results indicate that Aqp1aa might have a greater physiological role in ammonia excretion than in osmoregulation in A. testudineus.     

Acidification of the young phagosomes ofParamecium is mediated by proton pumps derived from the acidosomes

Summary Although it is generally accepted that phagosome acidification is induced through the activity of a vacuolar proton pump (V-ATPase) present on the phagosome membrane, exactly how these pumps are delivered to the phagosomes is not well understood. To study this question inParamecium, it was necessary to first show that an authentic V-ATPase was present on their phagosomal membranes. Three antibodies raised against V-ATPases or their subunits were each found to label one or two large digestive vacuoles (DVs) inParamecium multimicronucleatum when immunofluorescence microscopy was used. Using horseradish peroxidase immunocytochemistry to increase sensitivity, about 10 DVs were shown to contain a V-ATPase. In high magnification images and cryoultramicrotomy these proton pumps were found to be located on the acidosomes, suggesting the vacuolar proton pumps on the DVs originate from the acidosomes. The authenticity of the V-ATPase was further confirmed by its sensitivity to cold temperature and to the V-ATPase specific inhibitor, concanamycin B, which at 10 nM doubled the t_1/2 for vacuole acidification. Thus, we conclude that (1) acidosomes and some DVs ofParamecium have a bona-fide concanamycin B-sensitive and cold-sensitive V-ATPase, (2) the V-ATPase is delivered to the young DVs during acidosome fusion, and (3) the V-ATPase is involved in vacuole acidification. Finally, we have now determined thatParamecium has two immunologically related V-ATPases that are involved in two very different functions, (1) the acidification of phagosomes and (2) fluid segregation in the contractile vacuole complexes.Abbreviations BS-FITC bovine serum albumin-fluorescein isothiocyanate - CVC contractile vacuole complex - DV-I to DV-IV digestive vacuole stages 1 to 4 - HRP horseradish peroxidase - V-ATPase vacuolar proton pump     

A functional aquaporin co-localizes with the vacuolar proton pyrophosphatase to acidocalcisomes and the contractile vacuole complex of Trypanosoma cruzi

We cloned an aquaporin gene from Trypanosoma cruzi (TcAQP) that encodes a protein of 231 amino acids, which is highly hydrophobic. The protein has six putative transmembrane domains and the two signature motifs asparagine-proline-alanine (NPA) which have been shown, in other aquaporins, to be involved in the formation of an aqueous channel spanning the bilayer. TcAQP was sensitive to endo H treatment, suggesting that the protein is N-glycosylated. Oocytes of Xenopus laevis expressing TcAQP swelled under hyposmotic conditions indicating water permeability, which was abolished after preincubating oocytes with very low concentrations of the AQP inhibitors HgCl(2) and AgNO(3). glycerol transport was detected. No Immunofluorescence microscopy of T. cruzi expressing GFP-TcAQP showed co-localization of TcAQP with the vacuolar proton pyrophosphatase (V-H(+)-PPase), a marker of acidocalcisomes. This localization was confirmed by Western blotting and immunofluorescence staining using polyclonal antibodies against a C-terminal peptide of TcAQP. In addition, there was a strong anterior labeling in a vacuole, close to the flagellar pocket, that was distinct from the acidocalcisomes and that was identified by immunogold electron microscopy as the contractile vacuole complex. Taking together, the presence of an aquaporin in acidocalcisomes and the contractile vacuole complex of T. cruzi, provides support for the role of these organelles in osmotic adaptations of these parasites.     

Monoclonal Antibody to a Bacterial Endonuclear Symbiont Holospora Cross Reacts with Proteins of Contractile Vacuole Radial Canals of Paramecium Species

ABSTRACT A monoclonal antibody (mAb) IR-2-1 was raised against a 67-kDa protein purified from the macronucleus-specific bacterial symbiont Holospora obtusa of Paramecium caudatum. The mAb was found to react with two bands (31 and 67-kDa) on gels of H. obtusa. Indirect immunofluorescence microscopy showed that these antigens were distributed inside the cells. However, unexpectedly, this mAb also cross reacted with the radial arms of the contractile vacuole in P. caudatum, P. tetraurelia, P. multimicronucleatum, P. jenningsi and P. bursaria as well as with their cytoplasm. Immunoelectron microscopy showed that the antigens were located on the decorated spongiome of the radial arms. In immunoblots, mAb IR-2-1 reacted with a band of 67 kDa in all Paramecium species examined. However, no band appeared in the immunoblot of isolated macronuclei of H. obtusa-free P. caudatum and no label was seen in the nuclear matrix of the macronucleus of air-dried P. caudatum. These results suggest that the 67-kDa antigen found in H. obtusa was not imported from the host macronucleus and the same antigen in the host contractile vacuoles and cytoplasm were not derived from the symbiont. These results also showed that an epitope on the decorated spongiome of the Paramecium species is shared by its bacterial symbiont. In contrast to the decorated tubule-specific mAb, DS-1, the antigens for IR-2-1 appeared to be loosely membrane bound as they were lost in paraformaldehyde fixed and acetone permeabilized Paramecium. Supplementary key words. Contractile vacuole complexes, Holospora obtusa, monoclonal antibody, Paramecium.     

红树林植物木榄水通道基因的克隆和表达

根据植物水通道基因保守区设计简并引物,采用RT-PCR方法,从木榄树叶中分离出水通道基因的cDNA片段;3′RACE获得3′端cDNA序列;再经5′RACE获得5′端部分cDNA序列,命名为PIP2,GenBank登录号为EF126757。该基因全长843个碱基,编码281个氨基酸,具有典型的植物水通道基因结构。该基因编码的蛋白质与含羞草(PIP2;5)、欧洲葡萄(PIP)、拟南芥(PIP3)等水通道蛋白的同源性分别为90%、91%、88%。Northern杂交分析表明,该基因在木榄树不同器官中的表达差异明显:根部有较高的表达水平,茎部较弱,而在叶中只能检测到微弱的信号。     

Yeast water channels: an overview of orthodox aquaporins

In yeast, the presence of orthodox aquaporins has been first recognized in Saccharomyces cerevisiae, in which two genes (AQY1 and AQY2) were shown to be related to mammal and plant water channels. The present review summarizes the putative orthodox aquaporin protein sequences found in available genomes of yeast and filamentous fungi. Among the 28 yeast genomes sequenced, most species present only one orthodox aquaporin, and no aquaporins were found in eight yeast species. Alignment of amino acid sequences reveals a very diverse group. Similarity values vary from 99% among species within the Saccharomyces genus to 34% between ScAqy1 and the aquaporin from Debaryomyces hansenii. All of the fungal aquaporins possess the known characteristic sequences, and residues involved in the water channel pore are highly conserved. Advances in the establishment of the structure are reviewed in relation to the mechanisms of selectivity, conductance and gating. In particular, the involvement of the protein cytosolic N‐terminus as a channel blocker preventing water flow is addressed. Methodologies used in the evaluation of aquaporin activity frequently involve the measurement of fast volume changes. Particular attention is paid to data analysis to obtain accurate membrane water permeability parameters. Although the presence of aquaporins clearly enhances membrane water permeability, the relevance of these ubiquitous water channels in yeast performance remains obscure.     

Immunohistochemical localization of aquaporin 10 in the apical membranes of the human ileum: a potential pathway for luminal water and small solute absorption

A new member of the aquaporin family (AQP10) has recently been identified in the human small intestine by molecular cloning and in situ hybridization. Ribonuclease protection assay and northern blotting have demonstrated that AQP10 is expressed in the human duodenum and jejunum. However, the subcellular distribution of the AQP10 protein and its plasma membrane polarization have not yet been established. The objective of this study was to determine the distribution of the AQP10 protein in the human ileum by immunohistochemistry and western blotting using a polyclonal antibody raised against a unique 17-amino acid peptide derived from the human AQP10 sequence. The distribution of the AQP1 and AQP3 proteins was also studied by immunohistochemical staining using affinity-purified polyclonal antibodies. Results revealed that the AQP10 protein is preferentially targeted to the apical membrane domain of absorptive intestinal epithelial cells, whereas AQP3 is located in the basolateral membrane of the cells and AQP1 expression is restricted to the mucosal microvascular endothelia. The presence of AQP10 in the apical membrane of intestinal villi suggests that this protein may represent an entry pathway for water and small solutes from the lumen across to the mucosal side.     

Membrane tubulation and proton pumps

Summary A model is presented for one type of membrane tubulation. In this model large transmembrane protein complexes link together to form helical bands. To form the helical bands the individual complexes within the band would need to form a slight bend and twist with adjacent complexes as they link together. Such linkages would lead to the band and lipid bilayer extending out from the plane of the membrane to form a tube of a diameter equal to or smaller than the radial diameter of the helix. This model is supported by two examples of membrane tubulation found inParamecium, in which proton pumps are the transmembrane complexes that are associated together to form the helically coiled bands. These include the helically linked F₀F₁ complexes of the mitochondrial inner membrane and the V₀V₁ complexes of the decorated tubules of the contractile vacuole complex. Such tubules result in very high surface-to-volume ratios and may be important in efficient ATP production or in forming proton gradients for transporting ions across membranes.     

Presence of aquaporin and V-ATPase on the contractile vacuole of Amoeba proteus.

BACKGROUND INFORMATION: The results of water permeability measurements suggest the presence of an AQP (aquaporin) in the membrane of the CV (contractile vacuole) in Amoeba proteus [Nishihara, Shimmen and Sonobe (2004) Cell Struct. Funct. 29, 85-90]. RESULTS: In the present study, we cloned an AQP gene from A. proteus [ApAQP (A. proteus AQP)] that encodes a 295-amino-acid protein. The protein has six putative TMs (transmembrane domains) and two NPA (Asn-Pro-Ala) motifs, which are conserved among various AQPs and are thought to be involved in the formation of water channels that span the lipid bilayer. Using Xenopus oocytes, we have demonstrated that the ApAQP protein product can function as a water channel. Immunofluorescence microscopy with anti-ApAQP antibody revealed that ApAQP is detected on the CV membrane and on the vesicles around the CV. The presence of V-ATPase (vacuolar H+-ATPase) on the vesicle membrane around the CV was also detected. CONCLUSIONS: Our data on ApAQP allow us to provide the first informed explanation of the high water permeability of the CV membrane in amoeba. Moreover, the results suggest that vesicles possessing V-ATPase are involved in generating an osmotic gradient. Based on our findings, we propose a new hypothesis for the mechanism of CV function.     

Membrane Dynamics of the Contractile Vacuole Complex of Paramecium1

ABSTRACT. Membrane dynamics of the contractile vacuole complex of Paramecium were investigated using conventional electron microscopy of cells so that the vacuoles were serial-sectioned longitudinally and transversely. During systole, vacuolar membrane collapses first into flattened cisternae which undergo further modification into a mass of interconnected small membrane tubules. These tubules retain their connections with the radiating microtubular ribbons; consequently they are found only in the poleward hemisphere. Permanent connections between ampullae and the collapsed vacuole membrane could not be verified nor was a sphincter-like mechanism for closing such a junction observed. Membranes of the ampullae and the collecting canals also collapse to varying extents into arrays of tubules that remain bound to microtubular ribbons during diastole. Thus vacuole, ampullae, and collecting canal membranes all assume tubular forms when internal volume is at a minimum. Having failed to observe a microfilamentous encasement of the vacuole, we suggest that an alternative mechanism for the “contractile” function should be sought. One such is based on fluid volume increase and fluid flow within transiently interconnected tubular membrane systems that cycle between a tubular and a planar membrane form as internal volume is periodically increased and reduced. The driving force for this mechanism might best be sought in the molecular structure of the membranes of the contractile vacuole complex.     

Calcium Regulation in the Protozoan Model,Paramecium tetraurelia

Early in eukaryotic evolution, the cell has evolved a considerable inventory of proteins engaged in the regulation of intracellular Ca²⁺ concentrations, not only to avoid toxic effects but beyond that to exploit the signaling capacity of Ca²⁺ by small changes in local concentration. Among protozoa, the ciliate Paramecium may now be one of the best analyzed models. Ciliary activity and exo‐/endocytosis are governed by Ca²⁺, the latter by Ca²⁺ mobilization from alveolar sacs and a superimposed store‐operated Ca²⁺‐influx. Paramecium cells possess plasma membrane‐ and endoplasmic reticulum‐resident Ca²⁺‐ATPases/pumps (PMCA, SERCA), a variety of Ca²⁺ influx channels, including mechanosensitive and voltage‐dependent channels in the plasma membrane, furthermore a plethora of Ca²⁺‐release channels (CRC) of the inositol 1,4,5‐trisphosphate and ryanodine receptor type in different compartments, notably the contractile vacuole complex and the alveolar sacs, as well as in vesicles participating in vesicular trafficking. Additional types of CRC probably also occur but they have not been identified at a molecular level as yet, as is the equivalent of synaptotagmin as a Ca²⁺ sensor for exocytosis. Among established targets and sensors of Ca²⁺ in Paramecium are calmodulin, calcineurin, as well as Ca²⁺/calmodulin‐dependent protein kinases, all with multiple functions. Thus, basic elements of Ca²⁺ signaling are available for Paramecium.     

Applying bimolecular fluorescence complementation to screen and purify aquaporin protein:protein complexes

Protein:protein interactions play key functional roles in the molecular machinery of the cell. A major challenge for structural biology is to gain high‐resolution structural insight into how membrane protein function is regulated by protein:protein interactions. To this end we present a method to express, detect, and purify stable membrane protein complexes that are suitable for further structural characterization. Our approach utilizes bimolecular fluorescence complementation (BiFC), whereby each protein of an interaction pair is fused to nonfluorescent fragments of yellow fluorescent protein (YFP) that combine and mature as the complex is formed. YFP thus facilitates the visualization of protein:protein interactions in vivo, stabilizes the assembled complex, and provides a fluorescent marker during purification. This technique is validated by observing the formation of stable homotetramers of human aquaporin 0 (AQP0). The method's broader applicability is demonstrated by visualizing the interactions of AQP0 and human aquaporin 1 (AQP1) with the cytoplasmic regulatory protein calmodulin (CaM). The dependence of the AQP0‐CaM complex on the AQP0 C‐terminus is also demonstrated since the C‐terminal truncated construct provides a negative control. This screening approach may therefore facilitate the production and purification of membrane protein:protein complexes for later structural studies by X‐ray crystallography or single particle electron microscopy.     

The Contractile Vacuole as a Key Regulator of Cellular Water Flow in Chlamydomonas reinhardtii

Most freshwater flagellates use contractile vacuoles (CVs) to expel excess water. We have used Chlamydomonas reinhardtii as a green model system to investigate CV function during adaptation to osmotic changes in culture medium. We show that the contractile vacuole in Chlamydomonas is regulated in two different ways. The size of the contractile vacuoles increases during cell growth, with the contraction interval strongly depending on the osmotic strength of the medium. In contrast, there are only small fluctuations in cytosolic osmolarity and plasma membrane permeability. Modeling of the CV membrane permeability indicates that only a small osmotic gradient is necessary for water flux into the CV, which most likely is facilitated by the aquaporin major intrinsic protein 1 (MIP1). We show that MIP1 is localized to the contractile vacuole, and that the expression rate and protein level of MIP1 exhibit only minor fluctuations under different osmotic conditions. In contrast, SEC6, a protein of the exocyst complex that is required for the water expulsion step, and a dynamin-like protein are upregulated under strong hypotonic conditions. The overexpression of a CreMIP1-GFP construct did not change the physiology of the CV. The functional implications of these results are discussed.     

Cloning and Expression of Four Aquaporin Homologs from the Chinese Black Sleeper (Bostrychus sinensis): The Effects of Salinity Acclimation

Several fish species are known to possess mechanisms that allow them to adapt to environments with different salinities. The aim of this study was to investigate the effects of salinity on the expression of aquaporins (aqp1a, aqp3a, aqp8a, and aqp9a) in the gills and intestines of Chinese black sleeper. After 30 days of acclimation, the expression of aqp1a, aqp3a, and aqp9a in the gills was significantly higher in fish transferred to 5 ppt than in those transferred to 40 ppt seawater, whereas aqp8 expression was lower. In contrast, aqp1a, aqp3a, and aqp8a expression in the intestines was higher in fish acclimated in 40 ppt than in those acclimated in 5 ppt. During abrupt salinity acclimation, the levels of aqp1a and aqp9a in the gills varied over time in fish acclimated in 5 ppt, but not in 40 ppt. The aqp3a levels in gills were higher in the 5 ppt group after 24 h than in the 40 ppt. The expression level of aqp8a in gills was higher in 40 ppt than in 5 ppt, except for that at 12 h. In the intestines, expression level of aqp1a and aqp8a were significantly upregulated from 12 to 48 h following acclimation in 40 ppt and aqp3a was higher in 40 ppt group than in 5 ppt, while aqp9a expression exhibited an opposite trend. These findings suggest that aqp1a, aqp3a, aqp8a and aqp9a may play a major osmoregulatory role in water transport in the gills and intestines during acclimation to different salinity environment.

     

Acidocalcisomes and the contractile vacuole complex are involved in osmoregulation in Trypanosoma cruzi

Trypanosoma cruzi, the etiologic agent of Chagas disease, resists extreme fluctuations in osmolarity during its life cycle. T. cruzi possesses a robust regulatory volume decrease mechanism that completely reverses cell swelling when submitted to hypo-osmotic stress. The efflux of amino acids and K+ release could account for only part for this volume reversal. In this work we demonstrate that swelling of acidocalcisomes mediated by an aquaporin and microtubule- and cyclic AMP-mediated fusion of acidocalcisomes to the contractile vacuole complex with translocation of this aquaporin and the resulting water movement are responsible for the volume reversal not accounted for by efflux of osmolytes. Contractile vacuole bladders were isolated by subcellular fractionation in iodixanol gradients, showed a high concentration of basic amino acids and inorganic phosphate, and were able to transport protons in the presence of ATP or pyrophosphate. Taken together, these results strongly support a role for acidocalcisomes and the contractile vacuole complex in osmoregulation and identify a functional role for aquaporin in protozoal osmoregulation.     

The δA isoform of calmodulin kinase II mediates pathological cardiac hypertrophy by interfering with the HDAC4-MEF2 signaling pathway

Aquaporin 0 (AQP0) is a lens-specific protein comprising more than 30% of lens membrane protein content and is a member of the aquaporin family. Water permeates through AQP0 much more slowly than other aquaporin family members, and other compounds, such as glycerol, also permeate AQP0. In the lens, ascorbic acid (AA) is found at high concentrations, protecting the lens from photochemical events such as photo-oxidation. The aim of the present study was to clarify the function of AQP0. Mouse fibroblast L-cells stably expressing AQP0 were established and incubated in medium containing AA, and intracellular AA levels were measured by high-performance liquid chromatography (HPLC) and 2,6-dichlorophenol-indophenol (DCPIP) analysis. Intracellular AA levels in AQP0-expressing cells quickly rose and reached saturation 10 min after incubation in medium containing 1000 μM AA. In contrast, AA levels in cells slowly decreased when AA was washed out from the medium. Cells overexpressing AQP0 increased the cellular uptake of AA in a time- and concentration-dependent manner. These data suggest that AA as well as water permeates AQP0.AQP0 expression on Xenopus oocyte membranes was achieved by the injection of AQP0 cRNA into oocytes that were incubated in medium containing AA. Intracellular AA levels were then measured by HPLC. AA uptake was demonstrated in the AQP0-expressing oocytes and was shown to quickly reach saturation. Intracellular AA concentration in oocytes increased in a time- and concentration-dependent manner.The data in the present study show that AA permeates AQP0, reveal the role of AQP0 in AA permeability ex vivo, and also indicate that there is a difference between the import and export of AA via AQP0. These findings suggest that AQP0 plays an important role in controlling lens AA content.