A Role for the Membrane in Regulating Chlamydomonas Flagellar Length

Flagellar assembly requires coordination between the assembly of axonemal proteins and the assembly of the flagellar membrane and membrane proteins. Fully grown steady-state Chlamydomonas flagella release flagellar vesicles from their tips and failure to resupply membrane should affect flagellar length. To study vesicle release, plasma and flagellar membrane surface proteins were vectorially pulse-labeled and flagella and vesicles were analyzed for biotinylated proteins. Based on the quantity of biotinylated proteins in purified vesicles, steady-state flagella appeared to shed a minimum of 16% of their surface membrane per hour, equivalent to a complete flagellar membrane being released every 6 hrs or less. Brefeldin-A destroyed Chlamydomonas Golgi, inhibited the secretory pathway, inhibited flagellar regeneration, and induced full-length flagella to disassemble within 6 hrs, consistent with flagellar disassembly being induced by a failure to resupply membrane. In contrast to membrane lipids, a pool of biotinylatable membrane proteins was identified that was sufficient to resupply flagella as they released vesicles for 6 hrs in the absence of protein synthesis and to support one and nearly two regenerations of flagella following amputation. These studies reveal the importance of the secretory pathway to assemble and maintain full-length flagella.     

Heterologous Expression of Tulip Petal Plasma Membrane Aquaporins in Pichia pastoris for Water Channel Analysis

Water channels formed by aquaporins (AQPs) play an important role in the control of water homeostasis in individual cells and in multicellular organisms. Plasma membrane intrinsic proteins (PIPs) constitute a subclass of plant AQPs. TgPIP2;1 and TgPIP2;2 from tulip petals are members of the PIP family. In this study, we overexpressed TgPIP2;1 and TgPIP2;2 in Pichia pastoris and monitored their water channel activity (WCA) either by an in vivo spheroplast-bursting assay performed after hypo-osmotic shock or by growth assay. Osmolarity, pH, and inhibitors of AQPs, protein kinases (PKs), and protein phosphatases (PPs) affect the WCA of heterologous AQPs in this expression system. The WCA of TgPIP2;2-expressing spheroplasts was affected by inhibitors of PKs and PPs, which indicates that the water channel of this homologue is regulated by phosphorylation in P. pastoris. From the results reported herein, we suggest that P. pastoris can be employed as a heterologous expression system to assay the WCA of PIPs and to monitor the AQP-mediated channel gating mechanism, and it can be developed to screen inhibitors/effectors of PIPs.The movement of water across cell membranes has long been thought to occur by free diffusion through the lipid bilayer. However, the discovery of the membrane protein CHIP28 in red blood cells has suggested the involvement of protein channels (29), and it is now well established that transmembrane water permeability is facilitated by aquaporins (AQPs), water channel proteins that are found in bacteria, fungi, plants, and animals (1, 7, 13, 24). AQPs contain six transmembrane α-helices and five connecting loops, and both the N and C termini are located in the cytosol. The monomers assemble into tetrameric complexes, with each monomer forming an individual water channel (11, 14, 24, 33). Apart from the exceptions of AQP11 and AQP12 from mice, as described by K. Ishibashi (15), AQPs have two signature Asn-Pro-Ala motifs, which are located in the second intracellular and the fifth extracellular loops, B and E.While 13 different AQPs have been identified in mammals (16), more than 33 AQP homologues have been discovered in plants (6, 17, 30). Plant AQPs fall into four subclasses: (i) the plasma membrane (PM) intrinsic proteins (PIPs), which are localized in the PM; (ii) the tonoplast intrinsic proteins (TIPs), which are localized in the vacuolar membranes; (iii) the nodulin-26-like intrinsic proteins; and (iv) the small basic intrinsic proteins (24). In Arabidopsis and maize, there are 13 PIPs, which can be divided further into two subfamilies, PIP1 and PIP2 (6, 17).The functions and mechanisms of regulation of plant AQPs have been extensively investigated (7, 13, 18, 24). There have been several reports on the water channel activity (WCA) of specific AQPs and their regulation by protein phosphorylation (3, 4, 8, 12, 18, 25, 32, 33). It has been shown that the WCA of the PIP2 member SoPIP2;1 from spinach is regulated by phosphorylation at two Ser residues (19, 33).The physiologically interesting temperature-dependent opening and closing of tulip (Tulipa gesneriana) petals occur concomitantly with water transport and are regulated by reversible phosphorylation of an undefined PIP (4, 5). Recently, four PIP homologues were isolated from tulip petals, and their WCAs have been analyzed by heterologous expression in Xenopus laevis oocytes (3). It has been shown that the tulip PIP TgPIP2;2 (DDBJ/EMBL/GenBank accession no. {type:entrez-nucleotide,attrs:{text:AB305617,term_id:196259707,term_text:AB305617}}AB305617) is ubiquitously expressed in all organs of the tulip and that TgPIP2;2 is the most likely of the TgPIP homologues to be modulated by the reversible phosphorylation that regulates transcellular water transport and mediates petal opening and closing (3, 4). However, while the members of the PIP2 subfamily are characterized as water channels (6), TgPIP2;1 (DDBJ/EMBL/GenBank accession no. {type:entrez-nucleotide,attrs:{text:AB305616,term_id:196259705,term_text:AB305616}}AB305616) shows no significant WCA in the oocyte expression system (3). There is growing interest in research on AQPs due to their crucial roles in the physiology of plants and animals (1, 16, 21-24, 26-28, 36). The assay of AQP channel activity is usually performed using either a X. laevis oocyte expression system (29) or a stopped-flow light-scattering spectrophotometer (35), both of which are not widely available. Furthermore, the complexity of these methods and requirement of expertise limit their high-throughput applications. In contrast, a Pichia pastoris expression system is simple to use, inexpensive, and feasible and can be used in high-throughput applications. Although a P. pastoris expression system has been shown to assay the WCA of a TIP (9), extensive research is necessary with other AQPs such as PIPs or AQPs present in intragranular membranes to establish whether this assay system can be used to characterize a water channel and study its regulation mechanisms. With this in view, in the study reported herein, TgPIP2;1 and TgPIP2;2 have been heterologously expressed in P. pastoris, and their WCAs have been assayed. The effects of several factors, such as osmolarity, pH, and inhibitors of protein kinases (PKs) and protein phosphatases (PPs), on the WCA of the recombinant P. pastoris have been investigated. Based on the results, we demonstrate that the P. pastoris heterologous expression system can be used to rapidly characterize PIP channels, to monitor the effects of mutations, and to score the effects of inhibitors and abiotic factors.     

大豆叶片质膜蛋白激酶的自身磷酸化反应

利用Ferrell和Martin（1991）设计的测定印迹在PVDF膜上的蛋白激酶活性方法研究大豆叶片质膜蛋白激酶自身磷酸化反应活性,结果表明：与Mg－ATP相比,Mn－ATP是更有效的57KD蛋白激酶自身磷酸化反应底物;钙离子可以促进该激酶的自身磷酸化反应活性,而且EGTA可以显著降低它在SDS电泳中的迁移率,说明57KD蛋白激酶为依赖于钙的蛋白激酶;预磷酸化反应实验证明57KD蛋白激酶具有多个自身磷酸化反应位点,其分子的自身磷酸化状态可调性暗示这一激酶可能具有重要的生理功能。     

The Role of the Plasma Membrane in the Response of Plant Roots to Aluminum Toxicity

Al³⁺, the predominant form of solubilized aluminum at pH values below 5.0, has been shown to exert a profound inhibitory effect on root elongation. Al is known to accumulate at the root apex. The plasma membrane represents the first potential target for Al toxicity, due to its pronounced binding to phospholipids. Al appears to alter both the structure and functions of the plasma membrane, and a great deal of research has been conducted concerning the interactions between Al and the plasma membrane. In this review, recent findings regarding the interactions between Al and the plasma membrane are described, specifically findings involving Al-induced alterations in the structure and function of the plasma membrane.Key Words: acid soil, aluminum, plasma membrane, tolerance, toxicity     

A New LxxxA Motif in the Transmembrane Helix3 of Maize Aquaporins Belonging to the Plasma Membrane Intrinsic Protein PIP2 Group Is Required for Their Trafficking to the Plasma Membrane

Aquaporins play important roles in maintaining plant water status under challenging environments. The regulation of aquaporin density in cell membranes is essential to control transcellular water flows. This work focuses on the maize (Zea mays) plasma membrane intrinsic protein (ZmPIP) aquaporin subfamily, which is divided into two sequence-related groups (ZmPIP1s and ZmPIP2s). When expressed alone in mesophyll protoplasts, ZmPIP2s are efficiently targeted to the plasma membrane, whereas ZmPIP1s are retained in the endoplasmic reticulum (ER). A protein domain-swapping approach was utilized to demonstrate that the transmembrane domain3 (TM3), together with the previously identified N-terminal ER export diacidic motif, account for the differential localization of these proteins. In addition to protoplasts, leaf epidermal cells transiently transformed by biolistic particle delivery were used to confirm and refine these results. By generating artificial proteins consisting of a single transmembrane domain, we demonstrated that the TM3 of ZmPIP1;2 or ZmPIP2;5 discriminates between ER and plasma membrane localization, respectively. More specifically, a new LxxxA motif in the TM3 of ZmPIP2;5, which is highly conserved in plant PIP2s, was shown to regulate its anterograde routing along the secretory pathway, particularly its export from the ER.Aquaporins are of major importance to plant physiology, being essential for the regulation of transcellular water movement during growth and development (Maurel et al., 2008; Gomes et al., 2009; Heinen et al., 2009; Prado and Maurel, 2013; Chaumont and Tyerman, 2014). Aquaporins are small membrane proteins consisting of six transmembrane (TM) domains connected by five loops (A–E), and N and C termini facing the cytosol (Fig. 1A). They assemble as homotetramers and/or heterotetramers in the membrane, with each monomer acting as an independent water channel (Murata et al., 2000; Fetter et al., 2004; Gomes et al., 2009). Aquaporins form a highly divergent protein family in plants (Chaumont et al., 2001; Johanson et al., 2001), and this work focuses on the maize (Zea mays) plasma membrane intrinsic protein (ZmPIP) family (Chaumont et al., 2001). The regulation of the subcellular localization of these proteins is a key process controlling their density in the plasma membrane (PM) and, hence, their physiological roles (Hachez et al., 2013).Open in a separate windowFigure 1.Swapping TM3 of ZmPIP2;5 with that of ZmPIP1;2 retains the protein in intracellular structures. A, Cartoons representing the chimeric proteins composed of ZmPIP2;5, in which each TM has been replaced by the corresponding TM from ZmPIP1;2. All proteins are drawn with the cytosolic domains facing down. ZmPIP2;5 and ZmPIP1;2 portions are shown in black and white, respectively. All chimeras were fused to the C terminus of mYFP, which is not displayed for clarity purposes. B, Confocal microscopy images of maize mesophyll protoplasts transiently coexpressing mYFP-tagged ZmPIP2;5-PIP1;2 TM chimeric proteins (green) and the ER marker mCFP:HDEL (cyan). FM4-64 was added as a PM marker (red). Arrowheads in image 13 indicate accumulation of the protein in punctate structures that are not labeled by mCFP:HDEL. The localization patterns of the proteins of interest are representative of a total of at least 22 cells from three independent experiments. C, Confocal microscopy images of a maize mesophyll protoplast transiently expressing mYFP:ZmPIP2;5-TM3_PIP1;2 (green) and ST:mCFP (magenta). Arrowheads indicate colocalization in Golgi stacks. The images are representative of a total of 17 cells from two independent experiments. Bar = 5 µm.PIP aquaporins cluster in two groups (PIP1s and PIP2s), which are highly conserved across species (Kammerloher et al., 1994; Chaumont et al., 2000, 2001; Johanson et al., 2001; Anderberg et al., 2012). We previously showed that the maize PIP1 and PIP2 isoforms exhibit different water channel activities when expressed in Xenopus laevis oocytes, with only PIP2s increasing the membrane water permeability coefficient (P_f; Chaumont et al., 2000). However, when ZmPIP1 and ZmPIP2 are coexpressed, the isoforms physically interact to modify their stability and trafficking to the oocyte membrane, and synergistically increase the oocyte P_f (Fetter et al., 2004). Similar synergistic interactions between PIP1s and PIP2s have been reported in numerous plant species (Temmei et al., 2005; Mahdieh et al., 2008; Vandeleur et al., 2009; Bellati et al., 2010; Ayadi et al., 2011; Horie et al., 2011; Yaneff et al., 2014).PIPs were originally thought to be exclusively localized in the PM and were named accordingly (Kammerloher et al., 1994). However, recent experiments have shown that not all PIPs are located to the PM under all conditions, and that regulation of PIP subcellular localization is a highly dynamic process involving protein interactions (Boursiac et al., 2005, 2008; Zelazny et al., 2007, 2009; Uehlein et al., 2008; Besserer et al., 2012; Luu et al., 2012). When expressed singly in maize leaf mesophyll protoplasts, fluorescently tagged ZmPIP1s and ZmPIP2s differ in their subcellular localization. ZmPIP1s are retained in the endoplasmic reticulum (ER), whereas ZmPIP2s are targeted to the PM (Zelazny et al., 2007). However, upon coexpression, ZmPIP1s are relocalized from the ER to the PM, where they perfectly colocalize with ZmPIP2s. This relocalization results from their physical interaction as demonstrated by Förster resonance energy transfer/fluorescence lifetime imaging microscopy and immunoprecipitation experiments (Zelazny et al., 2007). These results indicate that ZmPIP2s, but not ZmPIP1s, possess signals that allow them to be delivered to the PM, and that hetero-oligomerization is required for ZmPIP1 trafficking to the PM. Interestingly, a diacidic motif (DxE, Asp-any amino acid-Glu) located in the N terminus of ZmPIP2;4, ZmPIP2;5, and Arabidopsis (Arabidopsis thaliana) AtPIP2;1 was shown to be required to exit the ER (Zelazny et al., 2009; Sorieul et al., 2011). Diacidic motifs interact with Secretory protein24, which is thought to be the main cargo-selection protein of the Coat proteinII complex that mediates vesicle formation at ER export sites (Miller et al., 2003). However, not all PM-localized PIP2s contain a diacidic ER export signal (Zelazny et al., 2009). In addition, swapping the N-terminal region of ER-retained ZmPIP1;2 with that of PM-localized ZmPIP2;5, which contains the functional diacidic motif, is not sufficient to trigger ER export of the protein (Zelazny et al., 2009). This result suggests that other export signals might be present in PIP2s and/or ER retention signals might be present in PIP1s elsewhere than in the N terminus.To identify new signals regulating ZmPIP1 and ZmPIP2 protein trafficking along the secretory pathway, we used a protein domain swapping-based approach and identified the TM3 as an important region that discriminates between ER-retained ZmPIP1;2 and PM-localized ZmPIP2;5. Specific mutations in the TM3 region of ZmPIP2;5 allowed the identification of a new ZmPIP2-conserved LxxxA motif, which regulates its export from the ER.     

水通道蛋白在塔里木兔肺组织中的表达和作用

通过检测塔里木兔Lepus yarcandensis肺脏中水通道蛋白(aquaporin,AQP)1、AQP3及AQP4的表达和分布情况,以探讨水通道蛋白在干旱区动物水液代谢中的作用。采用常规HE染色观察肺组织学结构,采用免疫组织化学检测AQP1、AQP3及AQP4在肺脏中的分布位置及表达。结果表明,AQP1分布在支气管上皮细胞,肺泡间质毛细血管内皮细胞以及肺泡上皮(Ⅰ、Ⅱ型)细胞。AQP3分布在小气管上皮顶质膜和大气道上皮细胞基底膜。AQP4分布在小气管和大气道上皮细胞基底膜。AQP1、AQP3及AQP4在肺中表达的强弱关系为AQP1>AQP4>AQP3。这些结果说明,水通道蛋白在塔里木兔肺泡腔、肺组织间隙及毛细血管腔之间水的转运中很可能起着很重要的作用。同时对吸入空气的湿润和呼出气体中水分的重吸收也具有重要意义。     

Changes in Stem and Leaf Hydraulics Preceding Leaf Shedding in Castanea Sativa L.

This paper describes changes in leaf water status and in stem, petiole and leaf blade hydraulics preceding leaf senescence and shedding in Castanea sativa L. (chestnut). Measurements of maximum diurnal leaf conductance to water vapour (g_L), minimum water potential (_L), hydraulic conductance per unit leaf surface area of stems (K_SL), petioles (K_PL) and leaf blades (K_LL) and number of functional conduits and inside diameter distribution were performed in June, September and October 1999. In September, still green leaves had undergone some dehydration as indicated by decreased g_L (by 75 %) and _L with respect to June. In the same time, K_SL decreased by 88 %, while K_PL and K_LL decreased by 50 % and 20 % of the conduits of stems and 10 % of the petioles (all belonging to the widest diameter range) were no longer functioning, causing a decrease in the theoretical flow by 82 % in stems and 27 % in petioles. Stem xylem blockage was apparently due to tyloses growing into conduits. We advance the hypothesis that the entire process of leaf shedding and winter rest may be initiated by extensive stem embolism occurring during the summer.     

Role of ARF4L in Recycling Between Endosomes and the Plasma Membrane

The human ADP-ribosylation factor-like protein, ARF4L is a member of the ARF family, which are small GTP-binding proteins that play significant roles in vesicle transport and protein secretion. However, little is known about the physiological roles of ARF4L. In this study, to understand the biological functions of ARF4L, we carried out immunocytochemical analysis of ARF4L molecules with mutations in the functional domains. ARF4L was shown to be distributed to the plasma membrane following binding to GTP (Q80L), and into endosomes following binding to GDP (T35N). Moreover, the inactive-form of ARF4L (T35N) causes localization of transferrin receptors to the endosomal compartment, while the active form (Q80L) causes transport to the plasma membrane. These findings indicate that ARF4L drive the transport of cargo protein and subsequent fusion of recycling vesicles with the plasma membrane for maintenance of the cell surface.     

Dual Role of Plasma Membrane Electron Transport Systems in Defense

Bcause oxidative stress is one of the main sources of severe cellular damage, cells have different defense weapons against reactive oxygen species. Ubiquitous plasma membrane redox systems play a role in defense against oxidative stress damage. On the other hand, a tightly controlled and localized production of reactive oxygen species by a plasma membrane NADPH oxidase can be used as a potent microbicidal weapon. This dual, prooxidant and antioxidant role of plasma membrane electron transport systems in defense is studied and discussed.     

6-BA延缓大豆叶片衰老的作用与膜蛋白磷酸化状态的关系

蛋白激酶（proteinkinase,PK）和蛋白磷酸酯酶（pIDt6inphOSpha～,PP）是生物体内催化蛋白质磷酸化／脱磷酸化过程的两种重要酶类。目前已有越来越多的实验证据表明：这种可逆的磷酸化／脱磷酸化过程所导致的蛋白质（酶）活性的改变是生物体内信号传导过程中的重要环节（Hunter1995）。已有一些实验系统涉及了植物激素对于植物蛋白磷酸化过程的影响（Mi－zogUchi等1994,Sano和Youssefian1994）,并有一些与此相关的蛋白激酶和蛋白磷酸酯酶的基因被克隆（kleber等1993,temp等1994）。细胞分裂素延缓植物叶片衰老的作用早已被各种实…     

Role of the Plasma Membrane H+-ATPase in K+ Transport

The role of the plant plasma membrane H+-ATPase in K+ uptake was examined using red beet (Beta vulgaris L.) plasma membrane vesicles and a partially purified preparation of the red beet plasma membrane H+-ATPase reconstituted in proteoliposomes and planar bilayers. For plasma membrane vesicles, ATP-dependent K+ efflux was only partially inhibited by 100 [mu]M vanadate or 10 [mu]M carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. However, full inhibition of ATP-dependent K+ efflux by these reagents occurred when the red beet plasma membrane H+-ATPase was partially purified and reconstituted in proteoliposomes. When reconstituted in a planar bilayer membrane, the current/voltage relationship for the plasma membrane H+-ATPase showed little effect of K+ gradients imposed across the bilayer membrane. When taken together, the results of this study demonstrate that the plant plasma membrane H+-ATPase does not mediate direct K+ transport chemically linked to ATP hydrolysis. Rather, this enzyme provides a driving force for cellular K+ uptake by secondary mechanisms, such as K+ channels or H+/K+ symporters. Although the presence of a small, protonophore-insensitive component of ATP-dependent K+ transport in a plasma membrane fraction might be mediated by an ATP-activated K+ channel, the possibility of direct K+ transport by other ATPases (i.e. K+-ATPases) associated with either the plasma membrane or other cellular membranes cannot be ruled out.     

Phosphate Transport across the Plasma Membrane of Wheat Leaf Protoplasts: Characteristics and Inhibitor Specificities

The kinetics and inhibitor specificities of phosphate transport across the plasma membrane of wheat leaf mesophyll protoplasts have been examined. Studies were also carried out on the effects of light and pH on phosphate transport and the plasma membrane electropotential. At pH 5.8 (30°C), protoplasts accumulated phosphate at the rate of 3.9 ± 0.2 nanomoles per milligram protein per hour. Phosphate uptake rates and inhibitor specificities for the leaf cell plasma membrane phosphate transporter were qualitatively similar to those observed with root protoplasts. Neither picrylsulfonic acid, or p-chloromercuribenzene sulfonate affected phosphate uptake significantly at 0.1 millimolar. Of all compounds tested, carbonyl cyanide-p-trifluoromethoxy phenylhydrazone was the most effective inhibitor of phosphate uptake (60% at 0.1 millimolar). Tribenzylphosphate inhibited uptake by 34% while dibenzylphosphate had no effect. The plasma membrane electropotential was found to be −37 ± 3 millivolts. Initiation of photosynthesis lowered the membrane potential to −39 ± 3 millivolts. Inhibition of phosphate uptake by 34% with the substrate analog tribenzylphosphate resulted in a measured membrane potential of −33 ± 3 millivolts. These changes in potential were not significant at the 5% probability level. Phosphate uptake rates remained constant under photosynthetic and nonphotosynthetic conditions. The utility of tribenzylphosphate as an inhibitor in plant systems is demonstrated.     

Salinity Effects on the Activity of Plasma Membrane H+and Ca2+Transporters in Bean Leaf Mesophyll: Masking Role of the Cell Wall

Net fluxes of H⁺and Ca²⁺were measured in the mesophyll tissueof broad bean (Vicia faba L.) leaves and in protoplasts derivedfrom these cells. NaCl at 90 m M enhanced H⁺extrusion in bothprotoplasts and tissue, but in different ways. Proton extrusionwas inhibited by vanadate, suggesting the involvement of theplasma membrane H⁺-ATPase in cell responses to salinity. Therewas virtually no effect of NaCl on the net Ca²⁺flux in protoplasts,while in the tissue a large transient Ca²⁺efflux followed thesalt treatment. Salt-induced Ca²⁺efflux was essentially independentof external Ca²⁺concentrations in the range 0.1 to 10 m M. Also,Ca²⁺flux responses were ‘saturated’ above 50 m MNaCl. It is suggested that almost all the measured Ca²⁺fluxoriginates from Na⁺/Ca²⁺and H⁺/Ca²⁺ion exchange in the cellwall. This conclusion was supported by the results of modellingcation exchange in the cell wall. Copyright 2000 Annals of BotanyCompany Salinity, membrane transporters, wall ion exchange, proton, calcium, Vicia faba     

How Cholesterol Could Be Drawn to the Cytoplasmic Leaf of the Plasma Membrane by Phosphatidylethanolamine

In the mammalian plasma membrane, cholesterol can translocate rapidly between the exoplasmic and cytoplasmic leaves, so that its distribution between them should be given by the equality of its chemical potential in the leaves. Due to its favorable interaction with sphingomyelin, which is almost entirely in the outer leaf, one expects the great majority of cholesterol to be there also. Experimental results do not support this, implying that there is some mechanism attracting cholesterol to the inner leaf. We hypothesize that it is drawn there to reduce the bending free energy of the membrane caused by the presence of PE (phosphatidylethanolamine). It does this in two ways: first by simply diluting the amount of PE in the inner leaf, and second by ordering the tails of the PE to reduce its spontaneous curvature. Incorporating this mechanism into a model free energy for the bilayer, we find that between 50 and 60% of the total cholesterol should be in the inner leaf of human erythrocytes.     

Sugar-Induced Increase of Calcium-Dependent Protein Kinases Associated with the Plasma Membrane in Leaf Tissues of Tobacco

The sugar-inducible expression of genes for sporamin and [beta]-amylase in leaf explants of sweet potato (Ipomoea batatas) and that of a [beta]-glucuronidase-fusion gene, with the promoter of the gene for [beta]-amylase in leaves of tobacco (Nicotiana tabacum), requires Ca2+ signaling (M. Ohto, K. Hayashi, M. Isobe, K. Nakamura [1995] Plant J 7: 297-307), and it was inhibited by staurosporin and K252a, inhibitors of protein kinases. Autophosphorylation activities of several potential protein kinases in leaves of tobacco were significantly higher in younger leaves than in mature leaves. However, the autophosphorylation activities of these proteins in mature leaves, especially those of the major autophosphorylatable proteins with apparent molecular masses of 56 and 54 kD, increased upon treatment of leaf discs with a 0.3 M solution of sucrose, glucose, or fructose, did not increase with sorbitol or mannitol treatments, and the increase by sucrose was inhibited by cycloheximide. Autophosphorylation of the 56- and 54-kD protein in vitro was dependent on Ca2+ and inhibited by staurosporine, K-252a, and by W-7. These results suggest that they belong to the family of calcium-dependent protein kinases. They were concentrated in the plasma membrane fraction and were released from membrane vesicles by high salt or with sodium carbonate. The possible functions of these sugar-inducible calcium-dependent protein kinases associated with the plasma membrane are discussed.     

The Role of Membrane Lateral Tension in Calcium-Induced Membrane Fusion

Calcium-induced fusion of liposomes was studied with a view to understand the role of membrane tension in this process. Lipid mixing due to fusion was monitored by following fluorescence of rhodamine-phosphatidyl-ethanolamine incorporated into liposomal membrane at a self-quenching concentration. The extent of lipid mixing was found to depend on the rate of calcium addition: at slow rates it was significantly lower than when calcium was injected instantly. The vesicle inner volume was then made accessible to external calcium by adding calcium ionophore A23187. No effect on fusion was observed at high rates of calcium addition while at slow rates lipid mixing was eliminated. Fusion of labeled vesicles with a planar phospholipid membrane (BLM) was studied using fluorescence microscopy. Above a threshold concentration specific for each ion, Ca²⁺, Mg²⁺, Cd²⁺ and La³⁺ induce fusion of both charged and neutral membranes. The threshold calcium concentration required for fusion was found to be dependent on the vesicle charge, but not on the BLM charge. Pretreatment of vesicles with ionophore and calcium inhibited vesicle fusion with BLM. This effect was reversible: chelation of calcium prior to the application of vesicle to BLM completely restored their ability to fuse. These results support the hypothesis that tension in the outer monolayer of lipid vesicle is a primary reason for membrane destabilization promoting membrane fusion. How this may be a common mechanism for both purely lipidic and protein-mediated membrane fusion is discussed. Received: 27 September 1999/Revised: 22 March 2000