Inside-out plant plasma membrane vesicles of high purity obtained by aqueous two-phase partitioning

AbstractHighly purified plasma membranes obtained from leaves of sugar beet (Beta vulgaris L.) by aqueous two-phase partitioning were separated into two fractions by further phase partition steps. The vesicles partitioning to the interface showed an ATP-dependent H+-uptake (measured using the pH probe acridine orange) and a negligible K+,Mg2+-ATPase latency, while the vesicles partitioning in the upper phase showed only slow H+-uptake and a high ATPase latency on addition of Triton X-100. Based on these results the material at the interface is estimated to contain ∼90% sealed, inside-out vesicles, and the material in the upper phase ∼90% sealed, right-side-out vesicle

PIP kinases and their role in plant tip growing cells

Phosphatidylinositol (4,5) bisphosphate　[PtdIns(4,5)P2], is a signaling lipid involved in many important processes in animal cells such as cytoskeleton organization, intracellular vesicular trafficking, secretion, cell motility, regulation of ion channels, and nuclear signaling pathways. In the last years PtdIns(4,5)P2 and its synthesizing enzyme, phosphatidylinositol phosphate kinase (PIPK), has been intensively studied in plant cells, revealing a key role in the control of polar tip growth. Analysis of the PIPK members from Arabidopsis thaliana, Oryza sativa and Physcomitrella patens showed that they share some regulatory features with animal PIPKs but also exert plant-specific modes of regulation. This review aims at giving an overview on the PIPK family from Arabidopsis thaliana and Physcomitrella patens. Even though their basic structure, modes of activation and physiological role is evolutionary conserved, modules responsible for plasma membrane localization are distinct for different PIPKs, depending on differences in physiological and/ or developmental status of cells, such as polarized and nonpolarized

Characterisation of a plasma membrane-associated phospholipase A(2) activity increased in response to cold acclimation

We here demonstrate the presence of a plasma membrane-associated phospholipase A(2) (EC 3.1.1.4; PLA(2)) activity in spinach (Spinacia oleracea) leaves. The pH profile of the spinach plasma membrane PLA(2) activity revealed two peaks, one at pH 4.4 and one at pH 5.5. The activity at pH 5.5 had an absolute requirement of Ca2+, with full enzyme activity at 10mumol/L Ca2+. The Ca2+-dependent PLA(2) activity was both heat sensitive and stimulated by diacylglycerol, whereas ATP completely inhibited the activity. Thus, the spinach plasma membrane contains a Ca2+-dependent PLA(2) activity, which has not previously been characterised in plants. Cold acclimation of spinach resulted in a 2.2-fold higher plasma membrane PLA(2) activity whereas the plasma membrane phospholipase D activity remained unaffected. Taken together, our data suggest a role of PLA(2) in cold acclimation in plants

Lysophosphatidylcholine Stimulates ATP Dependent Proton Accumulation in Isolated Oat Root Plasma Membrane Vesicles

Lysophosphatidylcholine at concentrations of 30 micromolar stimulated the rate of MgATP-dependent H(+)-accumulation in oat (Avena sativa L. cv Rhiannon) root plasma membrane vesicles about 85% while the passive permeability of H(+) was unchanged. Activation was dependent on chain length, degree of saturation, and head group of the lysophospholipid. A H(+)-ATPase assay was developed that allowed the simultaneous measurement of proton pumping and ATPase activity in the same sample. ATP hydrolysis was also stimulated by lysophospholipids and showed the same lipid specificity, but stimulation was only about 25% at 30 micromolar. At higher concentrations of lysophosphatidylcholine the ATPase activity in a latency-free system could be stimulated about 150%. The enzymic properties of proton pumping and ATP hydrolysis were otherwise identical with respect to vanadate sensitivity, K(m) for ATP and pH optimum. The stimulatory effect of lysophospholipids suggests that these compounds could be part of the regulatory system for plant plasma membrane H(+)-ATPase activity in vivo

Is membrane occupation and recognition nexus domain functional in plant phosphatidylinositol phosphate kinases?

Phosphatidylinositol phosphate kinase (PIPK) catalyzes a key step controlling cellular contents of phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2], a critical intracellular messenger involved in vesicle trafficking and modulation of actin cytoskeleton and also a substrate of phospholipase C to produce the two intracellular messengers, diacylglycerol and inositol-1,4,5-trisphosphate. In addition to the conserved C-terminal PIPK catalytic domain, plant PIPKs contain a unique structural feature consisting of a repeat of membrane occupation and recognition nexus (MORN) motifs, called the MORN domain, in the N-terminal half. The MORN domain has previously been proposed to regulate plasma membrane localization and phosphatidic acid (PA)-inducible activation. Recently, the importance of the catalytic domain, but not the MORN domain, in these aspects was demonstrated. These conflicting data raise the question about the function of the MORN domain in plant PIPKs. We therefore performed analyses of PpPIPK1 from the moss Physcomitrella patens to elucidate the importance of the MORN domain in the control of enzymatic activity; however, we found no effect on either enzymatic activity or activation by PA. Taken together with our previous findings of lack of function in plasma membrane localization, there is no positive evidence indicating roles of the MORN domain in enzymatic and functional regulations of PpPIPK1. Therefore, further biochemical and reverse genetic analyses are necessary to understand the biological significance of the MORN domain in plant PIPKs

Identification of a novel calreticulin isoform (Crt2) in human and mouse

Calreticulin is a Ca2+-binding chaperone localized mainly in the endoplasmic/sarcoplasmic reticulum in all higher organisms. To date, only one calreticulin isoform has been identified in human and mouse. Here we report a novel calreticulin isoform (Crt2) in human and mouse, with 53 (human) and 49% (mouse) identity to the previously identified calreticulin in respective species. The gene encoding the novel human calreticulin isoform spans 17 kb of genomic DNA and is expressed in testis, showing a similar expression as the chaperone calmegin. Phylogenetic analysis shows that two or more calreticulin (crt) genes are present both in plants and in mammals. The duplication of the crt gene in human and mouse suggests functional diversity, and variations in expression patterns among calreticulins. Two novel calreticulin (Crt2) isoforms, with high homology to the human and mouse calreticulin isoform (Crt2), were also identified in pig and rat via expressed sequence tags. (C) 2002 Elsevier Science B.V. All rights reserved