Putative Plasma Membrane Research Articles

Dissecting sensor functions in cell wall integrity signaling in Kluyveromyces lactis

KlWSC1, KlWSC2/3 and KlMID2, which encode putative plasma membrane sensors for cell wall integrity signaling in Kluyveromyces lactis, were cloned and characterized. Double and triple deletion mutants show severe cell integrity defects, indicating overlapping functions. The Klwsc1 Klmid2 double deletion phenotype can be suppressed by overexpression of the downstream components KlROM2, KlPKC1 and KlBCK1. KlWsc1 sensor domain analyses showed that an amino-terminal elongation as well as an extension within the cytoplasmic domain are dispensable for function. Heterologous complementation by KlMID2 and KlWSC1 in Saccharomyces cerevisiae is only achieved upon overexpression. In contrast to ScMID2, ScWSC1 complements in K. lactis. Functional studies with chimeric Mid2 constructs indicate that species specificity is mainly conferred by the extracellular domain. Sensor-GFP fusions localize to the plasma membrane, with a cell cycle dependent distribution of KlWsc1-GFP. Both Wsc-type sensors concentrate in discrete spots within the plasma membrane.

The immunomodulatory protein SV‐IV protects serum‐deprived cells against apoptosis but not against G0/G1 arrest: Possible implications for the survival of implanting embryo

Serum deprivation induced in human lymphoblastoid Raji cells oxidative stress-associated apoptotic death and G0/G1 cell cycle arrest. Addition into culture medium of the immunomodulatory protein Seminal vesicle protein 4 (SV-IV) protected these cells against apoptosis but not against cycle arrest. The antiapoptotic activity was related to: (1) decrease of endocellular reactive Oxygen species (ROS) (2) increase of mRNAs encoding anti-oxidant enzymes (catalase, G6PD) and antiapoptotic proteins (survivin, cox-1, Hsp70, c-Fos); (3) decrease of mRNAs encoding proapoptotic proteins (c-myc, Bax, caspase-3, Apaf-1). The biochemical changes underlaying these effects were probably induced by a protein tyrosine kinase (PTK) activity triggered by the binding of SV-IV to its putative plasma membrane receptors. The ineffectiveness of SV-IV to abrogate the cycle arrest was accounted for by its downregulating effects on D1,3/E G1-cyclins and CdK2/4 gene expression, ppRb/pRb ratio, and intracellular ROS concentration. In conclusion, these experiments: (1) prove that SV-IV acts as a cell survival factor; (2) suggest the involvement of a PTK in SV-IV signaling; (3) point to cell cycle-linked enzyme inhibition as responsible for cycle arrest; (4) provide a model to dissect the cycle arrest and apoptosis induced by serum withdrawal; (5) imply a possible role of SV-IV in the survival of hemiallogenic implanting embryos.

AtNHX8, a member of the monovalent cation:proton antiporter‐1 family in Arabidopsis thaliana, encodes a putative Li+/H+ antiporter

The Arabidopsis monovalent cation:proton antiporter-1 (CPA1) family includes eight members, AtNHX1-8. AtNHX1 and AtNHX7/SOS1 have been well characterized as tonoplast and plasma membrane Na+/H+ antiporters, respectively. The proteins AtNHX2-6 have been phylogenetically linked to AtNHX1, while AtNHX8 appears to be related to AtNHX7/SOS1. Here we report functional characterization of AtNHX8. AtNHX8 T-DNA insertion mutants are hypersensitive to lithium ions (Li+) relative to wild-type plants, but not to the other metal ions such as sodium (Na+), potassium (K+) and caesium (Cs+). AtNHX8 overexpression in a triple-deletion yeast mutant AXT3 that exhibits defective Na+/Li+ transport specifically suppresses sensitivity to Li+, but does not affect Na+ sensitivity. Likewise, AtNHX8 overexpression complemented sensitivity to Li+, but not Na+, in sos1-1 mutant seedlings, and increased Li+ tolerance of both the sos1-1 mutant and wild-type seedlings. Results of Li+ and K+ measurement of loss-of-function and gain-of-function mutants indicate that AtNHX8 may be responsible for Li+ extrusion, and may be able to maintain K+ acquisition and intracellular ion homeostasis. Subcellular localization of the AtNHX8-enhanced green fluorescent protein (EGFP) fusion protein suggested that AtNHX8 protein is targeted to the plasma membrane. Taken together, our findings suggest that AtNHX8 encodes a putative plasma membrane Li+/H+ antiporter that functions in Li detoxification and ion homeostasis in Arabidopsis.

Crosstalk between L-type calcium channels and ZnT-1, a new player in rate-dependent cardiac electrical remodeling

Crosstalk between two membrane transport systems is an established mechanism underlying regulation. In this study, we investigated the interaction between ZnT-1, a putative plasma membrane zinc transporter, and L-type voltage-dependent calcium channels (LTCC). In the atrium of the myocardium decreased activity of the LTCC is a dominant feature of patients with atrial fibrillation. The trigger for this inhibition has been attributed to the rapid firing rates and consequent calcium overload in the atrial cardiomyocytes. However, the underlying mechanism of LTCC inhibition is still to be elucidated. Here, we showed that the expression of ZnT-1 inhibits the activity of L-type channels during electrical remodeling induced by rapid pacing. (i) Direct manipulations of ZnT-1 expression in cultured cardiomyocytes either by ZnT-1 overexpression or by ZnT-1 silencing with siRNA, decreased or enhanced, respectively, the barium influx through the LTCC. (ii) Co-expression of ZnT-1 with LTCC in Xenopus oocytes decreased whole cell barium current through LTCC. (iii) Rapid pacing of cultured cardiomyocytes (4 h, 100 ms cycle) increased ZnT-1 protein expression and inhibited the voltage-dependent divalent cation influx through the LTCC. Moreover, silencing ZnT-1 with siRNA prevented the rapid pacing induced inhibition of the LTCC (iv) Atrial pacing of anesthetized adult rats (4 h, 50 ms cycle) led to a significant increase in atrial ZnT-1 protein expression in parallel with the typical decrease of the refractory period in the atria. Taken together, these findings demonstrate that crosstalk between ZnT-1 and the L-type calcium channels may underlie atrial response to rapid pacing, suggesting that ZnT-1 is a significant participant in rate-dependent cardiac electrical remodeling.

Saccharomyces cerevisiae Multidrug Resistance Transporter Qdr2 Is Implicated in Potassium Uptake, Providing a Physiological Advantage to Quinidine-Stressed Cells

The QDR2 gene of Saccharomyces cerevisiae encodes a putative plasma membrane drug:H(+) antiporter that confers resistance against quinidine, barban, bleomycin, and cisplatin. This work provides experimental evidence of defective K(+) (Rb(+)) uptake in the absence of QDR2. The direct involvement of Qdr2p in K(+) uptake is reinforced by the fact that increased K(+) (Rb(+)) uptake due to QDR2 expression is independent of the Trk1p/Trk2p system. QDR2 expression confers a physiological advantage for the yeast cell during the onset of K(+) limited growth, due either to a limiting level of K(+) in the growth medium or to the presence of quinidine. This drug decreases the K(+) uptake rate and K(+) accumulation in the yeast cell, especially in the Deltaqdr2 mutant. Qdr2p also helps to sustain the decrease of intracellular pH in quinidine-stressed cells in growth medium at pH 5.5 by indirectly promoting H(+) extrusion affected by the drug. The results are consistent with the hypothesis that Qdr2p may also couple K(+) movement with substrate export, presumably with quinidine. Other clues to the biological role of QDR2 in the yeast cell come from two additional lines of experimental evidence. First, QDR2 transcription is activated under nitrogen (NH(4)(+)) limitation or when the auxotrophic strain examined enters stationary phase due to leucine limitation, this regulation being dependent on general amino acid control by Gcn4p. Second, the amino acid pool is higher in Deltaqdr2 cells than in wild-type cells, indicating that QDR2 expression is, directly or indirectly, involved in amino acid homeostasis.

Isolation by suppression-subtractive hybridization of genes preferentially expressed during early and late fiber development stages in cotton

As a main natural fiber source, cotton plays an important role in human life. To identify genes preferentially expressed during early and late cotton fiber development, we constructed two fiber subtracted libraries on the basis of PCR-selected subtraction using a pool of nonfiber tissues as the same driver and 10 days postanthesis (DPA) and 20 DPA fiber cells as testers, respectively. Through differential screening, 292 clones in both libraries were identified as being preferentially expressed during fiber development. Sequence analysis showed that 31 unique sequences were found in the library of 10 DPA fibers and 48 unique sequences were obtained in the library of 20 DPA fibers. In addition, there were 13 unique clones in common in both libraries. Many previously reported cotton fiber-related genes were included in both libraries. Northern hybridization was performed to further confirm the differential expression and to determine the pattern of mRNA accumulation of selected clones. As a result, all selected cDNAs showed highly preferential expression in developing cotton fibers. Four genes-putative gibberellin-regulated protein, putative tonoplast intrinsic protein, putative plasma membrane intrinsic protein, and Gossypium hirsutum putative membrane protein-were identified in 10 DPA fiber subtracted library, and they were found to be expressed a lot during early fiber development. On the other hand, those genes screened out of 20 DPA fiber subtracted library, like arabinogalactan protein and fiber glycosyl hydrolase family 19 protein, were found highly expressed in fibers with the maximal transcription level during developmental switch from elongation to cellulose deposition. By subtraction between fibers and five nonfiber tissues, two sets of genes were identified, and their fiber-specific or fiber-preferential expression indicated that they are involved in the network that controls cotton fiber development.

Vitamin D3—ein Prodrug verschiedener D3-Hormone

Vitamin D (Colecalciferol) ist wahrend des gesamten Lebens fur die Knochengesundheit von auserordentlicher Bedeutung. Vitamin D wird konsekutiv in der Leber in 25-Hydroxy-colecalciferol sowie in der Niere in 1α,25-25-Dihydroxy-colecalciferol (Calcitriol) uberfuhrt. Calcitriol ist das dominierende D3-Hormon, das eine grose Anzahl biologischer Effekte durch Angriff am klassischen nuklearen Vitamin-D-Rezeptor (VDRnuc) induziert. Es reguliert die Gentranskription in uber 30 Zielorganen. Calcitriol interagiert auch mit einem putativ zellmembranstandigen Rezeptor (VDRmem1,25) der fur schnelle biologische Antworten verantwortlich ist. 24R,25-Dihydroxy-colecalciferol ist als ein weiteres Hormon fur die Frakturheilung von essentieller Bedeutung. Es entfaltet seine biologischen Effekte durch stereospezifischen Angriff an einem zweiten membranstandigen Rezeptor, VDRmem24,25 Ein weiteres D3-Hormon, das fur die Regulation der Zellproliferation in der Prostata von zentraler Bedeutung ist, ist 25-Hydroxy-colecalciferol. Es wirkt hauptsachlich durch direkten Angriff am nuklearen Vitamin-D-Rezeptor, z. T. jedoch auch durch seine 1α-Hydroxylierung in der Prostata. Altere Menschen haben haufig wahrend des gesamten Jahres einen Mangel an 25-Hydroxy-colecalciferol. Eine Supplementierung mit taglich 400 IE Vitamin D ist erforderlich, um adaquate 25-Hydroxy-colecalciferol-Spiegel zu erzielen.

Phytophthora nicotianae PnPMA1 encodes an atypical plasma membrane H +-ATPase that is functional in yeast and developmentally regulated

PnPMA1, a gene encoding a putative P-type plasma membrane H +-ATPase, has been isolated by differential screening of a Phytophthora nicotianae germinated cyst cDNA library. PnPMA1 is differentially expressed during pathogen asexual development with a more than 10-fold increase in expression in germinated cysts, the stage at which plant infection is initiated, compared to vegetative or sporulating hyphae or motile zoospores. PnPMA1 proteins are encoded by two closely linked genes that have no introns and encode identical proteins having 1068 amino acid residues and a molecular mass of 116.3 kDa. PnPMA1 shows moderate identity (30–50%) to plant and fungal plasma membrane H +-ATPases and weak identity to other P-type cation-transporting ATPases. PnPMA1 contains all the catalytic domains characteristic of H +-ATPases but also has a distinct domain of ∼155 amino acids that forms a putative cytoplasmic loop between transmembrane domains 8 and 9, a feature that is not present in PMA1 proteins from other organisms. Polyclonal antibodies raised against the 155 residue domain were shown by immunogold labelling to react with a protein in the plasma membrane of P. nicotianae germinated cysts but not with the plasma membrane of motile zoospores. Genetic complementation experiments demonstrated that the P. nicotianae PnPMA1 is functional in yeast, Saccharomyces cerevisiae.

Interaction of the epithelial Ca2+ channels TRPV5 and TRPV6 with the intestine- and kidney-enriched PDZ protein NHERF4

The epithelial Ca(2+) channels TRPV5 and TRPV6 constitute the apical Ca(2+) influx pathway in epithelial Ca(2+) transport. PDZ proteins have been demonstrated to play a crucial role in the targeting or anchoring of ion channels and transporters in the apical domain of the cell. In this study, we describe the identification of NHERF4 (Na-P(i) Cap2/IKEPP/PDZK2) as a novel TRPV5- and TRPV6-associated PDZ protein. NHERF4 was identified using two separate yeast two-hybrid screens with the carboxyl termini of TRPV5 and TRPV6 as bait. Binding of the carboxyl termini of TRPV5 and TRPV6 with NHERF4 was confirmed by GST pull-down assays using in-vitro-translated NHERF4 or lysates of Xenopus laevis oocytes expressing NHERF4. Furthermore, the interaction was confirmed by GST pull-down and co-immunoprecipitation assays using in-vitro-translated full-length TRPV5 and Xenopus oocytes or HEK293 cells co-expressing NHERF4 and TRPV5/TRPV6, respectively. The fourth PDZ domain of NHERF4 was sufficient for the interaction, although PDZ domain 1 also contributed to the binding. The binding site for NHERF4 localized in a conserved region in the carboxyl terminus of TRPV5 and was distinct from the binding site of the PDZ protein NHERF2. NHERF4 predominantly localized at the plasma membrane of X. laevis oocytes and HeLa cells. This localization was independent of the presence of TRPV5. Therefore, we hypothesize a role for this novel PDZ protein as a putative plasma membrane scaffold for the epithelial Ca(2+) channels.

Isolation and characterization of a new Na+/H+antiporter geneOsNHA1from rice (Oryza sativaL.)

The full-length cDNA (3612 bp) of OsNHA1 was cloned by RT-PCR approach from rice (Oryza sativa L.), which encodes a putative plasma membrane Na+/H+ antiporter. Its deduced protein, OsNHA1, has 11 transmembrane domains and a significant similarity to a plasma membrane Na+/H+ antiporter AtNHA1 from Arabidopsis thaliana. Phylogenetic analysis showed that the OsNHA1 clusters with the plasma membrane Na+/H+ antiporters from various organisms. The semi-quantitative RT-PCR assay revealed that the expression of OsNHA1 was up-regulated in both shoots and roots of rice seedlings under salt stress, whereas it was not induced in the rice seedlings treated by drought stress.

Cloning, expression, purification, and properties of a putative plasma membrane hexokinase from Solanum chacoense

A full-length hexokinase cDNA was cloned from Solanum chacoense, a wild relative of the cultivated potato. Analysis of the predicted primary sequence suggested that the protein product, ScHK2, may be targeted to the secretory pathway and inserted in the plant plasma membrane, facing the cytosol. ScHK2 was expressed as a hexahistidine-tagged protein in Escherichia coli. Expression conditions for this construct were optimized using a specific anti-hexokinase polyclonal anti-serum raised against a truncated version of ScHK2. The full-length recombinant protein was purified to electrophoretic homogeneity using immobilized metal ion affinity chromatography followed by anion exchange chromatography on Fractogel EMD DEAE-650 (S). The purified enzyme had a specific activity of 5.3 μmol/min/mg protein. Its apparent K ms for glucose (23 μM), mannose (30 μM), fructose (5.2 mM), and ATP (61 μM) were in good agreement with values found in the literature for other plant hexokinases. Hexahistidine-tagged ScHK2 was highly sensitive to pH variations between 7.7 and 8.7. It was inhibited by ADP and insensitive to glucose-6-phosphate. These findings constitute the first kinetic characterization of a homogeneous plant hexokinase preparation. The relevance of ScHK2 kinetic properties is discussed in relation to the regulation of hexose metabolism in plants.

Isolation, cloning and expression analysis of EcPMA1, a putative plasma membrane H +-ATPase transporter gene from the biotrophic pathogenic fungus Erysiphe cichoracearum

Little is known at the molecular level about the transporters involved in nutrient transfer in the plant/powdery mildew interaction. A PCR-based approach was used to identify and isolate a partial-length cDNA coding for an isoform of the plasma membrane H +-ATPase ( EcPMA1) in the biotrophic pathogenic fungus Erysiphe cichoracearum. Southern analysis suggests that EcPMA1 exists as a single-copy gene. Sequence analysis indicated a high similarity of EcPMA1 to other fungal H +-ATPases. Expression of EcPMA1 increases in infected Arabidopsis leaves as the disease progresses, correlating with the growth of the pathogen.

A Putative Plasma Membrane Cation/proton Antiporter from Soybean Confers Salt Tolerance in Arabidopsis

Cation transport is thought to be an important process for ion homeostasis in plant cells. Here, we report that a soybean putative cation/proton antiporter GmCAX1 may be a mediator of this process. GmCAX1 is expressed in all tissues of the soybean plants but at a lower level in roots. Its expression was induced by PEG, ABA, Ca(2+), Na(+) and Li(+) treatments. The GmCAX1-GFP fusion protein was mainly localized in plasma membrane of the transgenic Arabidopsis plant cells and onion epidermal cells. Transgenic Arabidopsis plants overexpressing GmCAX1 accumulated less Na(+), K(+), and Li(+), and were more tolerant to elevated Li(+) and Na(+) levels during germination when compared with the controls. These results suggest that GmCAX1 may function as an antiporter for Na(+), K(+) and Li(+). Modulation of this antiporter may be beneficial for regulation of ion homeostasis and thus plant salt tolerance.

Molecular characterization of a cold-induced plasma membrane protein gene from wheat

As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Deltapmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat.

Ability of Sit4p To Promote K+Efflux via Nha1p Is Modulated by Sap155p and Sap185p

We demonstrate here that SAP155 encodes a negative modulator of K+ efflux in the yeast Saccharomyces cerevisiae. Overexpression of SAP155 decreases efflux, whereas deletion increases efflux. In contrast, a homolog of SAP155, called SAP185, encodes a positive modulator of K+ efflux: overexpression of SAP185 increases efflux, whereas deletion decreases efflux. Two other homologs, SAP4 and SAP190, are without effect on K+ homeostasis. Both SAP155 and SAP185 require the presence of SIT4 for function, which encodes a PP2A-like phosphatase important for the G1-S transition through the cell cycle. Overexpression of either the outwardly rectifying K+ channel, Tok1p, or the putative plasma membrane K+/H+ antiporter, Kha1p, increases efflux in both wild-type and sit4Delta strains. However, overexpression of the Na+-K+/H+ antiporter, Nha1p, is without effect in a sit4Delta strain, suggesting that Sit4p signals to Nha1p. In summary, the combined activities of Sap155p and Sap185p appear to control the function of Nha1p in K+ homeostasis via Sit4p.

Metastasis-associated Protein S100A4 Induces Angiogenesis through Interaction with Annexin II and Accelerated Plasmin Formation

Many advanced tumors overexpress and secrete the S100A4 protein that is known to promote angiogenesis and metastasis development. The mechanisms of this effect and the endothelial receptor for S100A4 are both still unknown. Here we report that extracellular S100A4 interacts with annexin II, an endothelial plasminogen co-receptor. Co-localization and direct binding of S100A4 and annexin II were demonstrated, and the binding site was identified in the N-terminal region of annexin II. S100A4 alone or in a complex with annexin II accelerated tissue plasminogen activator-mediated plasminogen activation in solution and on the endothelial cell surface through interaction of the S100A4 C-terminal lysines with the lysine-binding domains of plasminogen. A synthetic peptide corresponding to the N terminus of annexin II prevented S100A4-induced plasmin formation in the endothelial cell culture. Local plasmin formation induced by circulating S100A4 could contribute to tumor-induced angiogenesis and metastasis formation that makes this protein an attractive target for new anti-cancer and anti-angiogenic therapies.

Physiological characterization of Saccharomyces cerevisiae kha1 deletion mutants

Maintenance of intracellular K+ homeostasis is one of the crucial requisites for the survival of yeast cells. In Saccharomyces cerevisiae, the high K+ content corresponds to a steady state between simultaneous influx and efflux across the plasma membrane. One of the transporters formerly believed to extrude K+ from the yeast cells (besides Ena1-4p and Nha1p) was named Kha1p and presumed as a putative plasma membrane K+/H+ antiporter. We prepared kha1 and tok1-kha1 deletion strains in the B31 and MAB 2d background. Both the strains contain the ena1-4 and nha1 deletions; that means they lack the main active sodium and potassium efflux systems. MAB 2d has additional trk1 and trk2 deletions, i.e. is impaired in active K+ uptake as well. We performed a large physiological study with these strains to specify the phenotype of kha1 deletion. In our experiments, no difference in K+ content or efflux was observed in strains lacking the KHA1 gene compared with control strains. Two main phenotype manifestations of the kha1 deletion were growth defect on high external pH and hygromycin sensitivity. The correlation between these phenotypes and the kha1 deletion was confirmed by plasmid complementation. Fluorescence microscopy of green fluorescent protein (GFP)-tagged Kha1p showed that this antiporter is localized preferentially intracellularly (in contrast to the plasma membrane Na+/H+ antiporter Nha1p). Based on these findings, Kha1p is probably not localized in plasma membrane and does not mediate efflux of alkali metal cations from cells, but is important for the regulation of intracellular cation homeostasis and optimal pH control, similarly as the Nhx1p.

Arabidopsis Plasma Membrane Proteomics Identifies Components of Transport, Signal Transduction and Membrane Trafficking

In order to identify integral proteins and peripheral proteins associated with the plasma membrane, highly purified Arabidopsis plasma membranes from green tissue (leaves and petioles) were analyzed by mass spectrometry. Plasma membranes were isolated by aqueous two-phase partitioning, which yields plasma membrane vesicles with a cytoplasmic-side-in orientation and with a purity of 95%. These vesicles were turned inside-out by treatment with Brij 58 to remove soluble contaminating proteins enclosed in the vesicles and to remove loosely bound contaminating proteins. In total, 238 putative plasma membrane proteins were identified, of which 114 are predicted to have transmembrane domains or to be glycosyl phosphatidylinositol anchored. About two-thirds of the identified integral proteins have not previously been shown to be plasma membrane proteins. Of the 238 identified proteins, 76% could be classified according to function. Major classes are proteins involved in transport (17%), signal transduction (16%), membrane trafficking (9%) and stress responses (9%). Almost a quarter of the proteins identified in the present study are functionally unclassified and more than half of these are predicted to be integral.

Protein complexes of the plant plasma membrane resolved by Blue Native PAGE

With the characterization of the total genomes of Arabidopsis thaliana and Oryza sativa, several putative plasma membrane components have been identified. However, a lack of knowledge at the protein level, especially for hydrophobic proteins, have hampered analyses of physiological changes. To address whether protein complexes may be present in the native membrane, we subjected plasma membranes isolated from Spinacia oleracea leaves to blue‐native polyacrylamide gel electrophoresis (BN‐PAGE). BN‐PAGE is well established in the separation of functional membrane protein complexes from mitochondria and chloroplasts, but a resolved protein complex pattern from PM of eukaryotic cells has previously not been reported. Using this method, protein complexes from Spinacia oleracea PM could be efficiently solubilized and separated, including the highly hydrophobic aquaporin (apparent molecular mass 230 kDa), a putative tetramer of H+‐ATPase, and several less abundant complexes with apparent masses around or above 750 kDa. After denaturation and separation of the complexes into their subunits in a second dimension (SDS‐PAGE), several of the complexes were identified as hydrophobic membrane proteins. Large amounts of protein (up to 1 mg) can be resolved in each lane, which suggests that the method could be used to study also low‐abundance protein complexes, e.g. under different physiological conditions.

Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals.

The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.