Ion transport, membrane traffic and cellular volume control

Throughout their development, plants balance cell surface area and volume with ion transport and turgor. This balance lies at the core of cellular homeostatic networks and is central to the capacity to withstand abiotic as well as biotic stress. Remarkably, very little is known of its mechanics, notably how membrane traffic is coupled with osmotic solute transport and its control. Here we outline recent developments in the understanding of so-called SNARE proteins that form part of the machinery for membrane vesicle traffic in all eukaryotes. We focus on SNAREs active at the plasma membrane and the evidence for specialisation in enhanced, homeostatic and stress-related traffic. Recent studies have placed a canonical SNARE complex associated with the plasma membrane in pathogen defense, and the discovery of the first SNARE as a binding partner with ion channels has demonstrated a fundamental link to inorganic osmotic solute uptake. Work localising the channel binding site has now identified a new and previously uncharacterised motif, yielding important clues to a plausible mechanism coupling traffic and transport. We examine the evidence that this physical interaction serves to balance enhanced osmotic solute uptake with membrane expansion through mutual control of the two processes. We calculate that even during rapid cell expansion only a minute fraction of SNAREs present at the membrane need be engaged in vesicle traffic at any one time, a number surprisingly close to the known density of ion channels at the plant plasma membrane. Finally, we suggest a framework of alternative models coupling transport and traffic, and approachable through direct, experimental testing.     

A novel motif essential for SNARE interaction with the K(+) channel KC1 and channel gating in Arabidopsis

The SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) protein SYP121 (=SYR1/PEN1) of Arabidopsis thaliana facilitates vesicle traffic, delivering ion channels and other cargo to the plasma membrane, and contributing to plant cell expansion and defense. Recently, we reported that SYP121 also interacts directly with the K(+) channel subunit KC1 and forms a tripartite complex with a second K(+) channel subunit, AKT1, to control channel gating and K(+) transport. Here, we report isolating a minimal sequence motif of SYP121 prerequisite for its interaction with KC1. We made use of yeast mating-based split-ubiquitin and in vivo bimolecular fluorescence complementation assays for protein-protein interaction and of expression and electrophysiological analysis. The results show that interaction of SYP121 with KC1 is associated with a novel FxRF motif uniquely situated within the first 12 residues of the SNARE sequence, that this motif is the minimal requirement for SNARE-dependent alterations in K(+) channel gating when heterologously expressed, and that rescue of KC1-associated K(+) current of the root epidermis in syp121 mutant Arabidopsis plants depends on expression of SNARE constructs incorporating this motif. These results establish the FxRF sequence as a previously unidentified motif required for SNARE-ion channel interactions and lead us to suggest a mechanistic framework for understanding the coordination of vesicle traffic with transmembrane ion transport.     

Turgor regulation in osmotically stressed Arabidopsis epidermal root cells. Direct support for the role of inorganic ion uptake as revealed by concurrent flux and cell turgor measurements

Hyperosmotic stress is known to significantly enhance net uptake of inorganic ions into plant cells. Direct evidence for cell turgor recovery via such a mechanism, however, is still lacking. In the present study, we performed concurrent measurements of net ion fluxes (with the noninvasive microelectrode ion flux estimation technique) and cell turgor changes (with the pressure-probe technique) to provide direct evidence that inorganic ion uptake regulates turgor in osmotically stressed Arabidopsis epidermal root cells. Immediately after onset of hyperosmotic stress (100/100 mM mannitol/sorbitol treatment), the cell turgor dropped from 0.65 to about 0.25 MPa. Turgor recovery started within 2 to 10 min after the treatment and was accompanied by a significant (30-80 nmol m-2 s-1) increase in uptake of K+, Cl-, and Na+ by root cells. In most cells, almost complete (>90% of initial values) recovery of the cell turgor was observed within 40 to 50 min after stress onset. In another set of experiments, we combined the voltage-clamp and the microelectrode ion flux estimation techniques to show that this process is, in part, mediated by voltage-gated K+ transporters at the cell plasma membrane. The possible physiological significance of these findings is discussed.     

Setting SNAREs in a different wood

Vesicle traffic is essential for cell homeostasis, growth and development in plants, as it is in other eukaryotes, and is facilitated by a superfamily of proteins known as soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs). Although SNAREs are well-conserved across phylla, genomic analysis for two model angiosperm species available to date, rice and Arabidopsis, highlights common patterns of divergence from other eukaryotes. These patterns are associated with the expansion of some gene subfamilies of SNAREs, the absence of others and the appearance of new proteins that show no significant homologies to SNAREs of mammals, yeast or Drosophila. Recent findings indicate that the functions of these plant SNAREs also extend beyond the conventional 'housekeeping' activities associated with vesicle trafficking. A number of SNAREs have been implicated in environmental responses as diverse as stomata movements and gravisensing as well as sensitivity to salt and drought. These proteins are essential for signal transduction and response and, in most cases, appear also to maintain additional roles in membrane trafficking. One common theme to this added functionality lies in control of non-SNARE proteins, notably ion channels. Other examples include interactions between the SNAREs and scaffolding or other structural components within the plant cell.     

Actin filaments and microtubules of Arabidopsis suspension cells show different responses to changing turgor pressure

Past decades have brought great advances in understanding the relationship between turgor pressure and plant cell growth. New studies have provided evidence that turgor pressure acts as a stimulus for cell growth, and is also a developmental cue for post-embryonic organogenesis. However, the subcellular mechanisms underlying plant cell turgor pressure sensing remain unclear. Here, using the relatively simple undifferentiated cells from suspension cultures, we report real-time in vivo observations of the reorganization of microtubules and actin microfilaments induced by turgor pressure changes. We found that these two cytoskeletal elements differed in their reorganization patterns. Our results will be useful in the understanding of the relationship between the cytoskeleton, turgor pressure, and stress in plant cell morphogenesis.     

Ptk2 contributes to osmoadaptation in the filamentous fungus Neurospora crassa

Hyphal tip-growing organisms often rely upon an internal hydrostatic pressure (turgor) to drive localized expansion of the cell. Regulation of the turgor in response to osmotic shock is mediated primarily by an osmotic MAP kinase cascade which activates osmolyte synthesis and ion uptake to effect turgor recovery. We characterized a Neurospora crassa homolog (PTK2) of ser/thr kinase regulators of ion transport in yeast to determine its role in turgor regulation in a filamentous fungi. The ptk2 mutant is osmosensitive, and has lower turgor poise than wildtype. The cause appears to be lower activity of the plasma membrane H⁺-ATPase. Its role in osmoadaptation is unrelated to the activity of the osmotic MAP kinase cascade. Instead, it acts in an alternative pathway that, like the osmotic MAP kinase cascade, also involves ion transport mediated osmoadaptation.     

Book reviews

The GLUT4 facilitative glucose transporter protein is primarily expressed in muscle and adipose tissue and accounts for the majority of post-prandial glucose uptake. In the basal or non-stimulated state, GLUT4 is localized to intracellular membrane compartments sequestered away from circulating glucose. However, in response to agonist stimulation, there is a marked redistribution of the GLUT4 protein to the cell surface membrane providing a transport route for the uptake of glucose. This GLUT4 translocation can be divided into four general steps: (i) GLUT4 vesicle trafficking outofthe storage pool, (ii) docking just below the cell surface, (iii) priming via the interactions of the SNARE proteins present on the vesicular and plasma membranes, and (iv) fusion of the GLUT4 vesicle with the plasma membrane. This review focuses on recent advances made in identification and characterization of the molecular events and protein interactions involved in these steps of insulin-stimulated GLUT4 translocation.     

Molecular machinery involved in the insulin-regulated fusion of GLUT4-containing vesicles with the plasma membrane (review).

The GLUT4 facilitative glucose transporter protein is primarily expressed in muscle and adipose tissue and accounts for the majority of post-prandial glucose uptake. In the basal or non-stimulated state, GLUT4 is localized to intracellular membrane compartments sequestered away from circulating glucose. However, in response to agonist stimulation, there is a marked redistribution of the GLUT4 protein to the cell surface membrane providing a transport route for the uptake of glucose. This GLUT4 translocation can be divided into four general steps: (i) GLUT4 vesicle trafficking out of the storage pool, (ii) docking just below the cell surface, (iii) priming via the interactions of the SNARE proteins present on the vesicular and plasma membranes, and (iv) fusion of the GLUT4 vesicle with the plasma membrane. This review focuses on recent advances made in identification and characterization of the molecular events and protein interactions involved in these steps of insulin-stimulated GLUT4 translocation.     

How do membrane proteins sense water stress?

Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmoprotectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift-activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein-lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift-activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins.     

Abscisic acid triggers the endocytosis of the arabidopsis KAT1 K+ channel and its recycling to the plasma membrane

Membrane vesicle traffic to and from the plasma membrane is essential for cellular homeostasis in all eukaryotes. In plants, constitutive traffic to and from the plasma membrane has been implicated in maintaining the population of integral plasma-membrane proteins and its adjustment to a variety of hormonal and environmental stimuli. However, direct evidence for evoked and selective traffic has been lacking. Here, we report that the hormone abscisic acid (ABA), which controls ion transport and transpiration in plants under water stress, triggers the selective endocytosis of the KAT1 K+ channel protein in epidermal and guard cells. Endocytosis of the K+ channel from the plasma membrane initiates in concert with changes in K+ channel activities evoked by ABA and leads to sequestration of the K+ channel within an endosomal membrane pool that recycles back to the plasma membrane over a period of hours. Selective K+ channel endocytosis, sequestration, and recycling demonstrates a tight and dynamic control of the population of K+ channels at the plasma membrane as part of a key plant signaling and response mechanism, and the observations point to a role for channel traffic in adaptive changes in the capacity for osmotic solute flux of stomatal guard cells.     

Functional conservation between yeast and plant endosomal Na(+)/H(+) antiporters

Vacuolar compartmentation of Na(+) is an essential mechanism for salinity tolerance since it lowers cytosolic Na(+) levels while contributing to osmotic adjustment for cell turgor and expansion. The AtNHX1 protein of Arabidopsis thaliana substituted functionally for ScNHX1, the endosomal Na(+)/H(+) antiporter of yeast. Ion tolerance conferred by AtNHX1 and ScNHX1 correlated with ion uptake into an intracellular pool that was energetically dependent on the vacuolar (H(+))ATPase. AtNHX1 localized to vacuolar membrane fractions of yeast. Hence, both transporters share an evolutionarily conserved function in Na(+) compartmentation. AtNHX1 mRNA levels were upregulated by ABA and NaCl treatment in leaf but not in root tissue.     

Plant 14-3-3 proteins assist ion channels and pumps

Turgor pressure is a cellular parameter, important for a range of physiological processes in plants, like cell elongation, gas exchange and gravitropic/phototropic bending. Regulation of turgor pressure involves ion and water transport at the expense of metabolic energy (ATP). The primary pump in the plasma membrane (the H(+)-ATPase) is a key player in turgor regulation since it provides the driving force for ion uptake, followed by water influx through osmosis. Using the phytotoxin fusicoccin (a well-known activator of the ATPase) as a tool, 14-3-3 proteins were identified as regulators of the H(+)-ATPase. Since fusicoccin has a dramatic effect on K(+) accumulation and cellular respiration as well, we studied whether 14-3-3 proteins play a role in the regulation of the mitochondrial F(0)F(1)-ATP synthase and ion channels in the vacuolar and plasma membranes. Besides the plasma membrane H(+)-ATPase, we have identified thus far at least four other transport proteins that are regulated by 14-3-3 proteins. The mechanism of regulation will be described and the possibility that 14-3-3 proteins act as coordinators of ion transporters with varied but interdependent functions will be discussed.     

Evidence on the mechanism of enhanced sucrose uptake at low cell turgor in leaf discs of Phaseolus coccinius

Plants often tolerate water deficits by lowering the osmotic potential of their cell sap. This may be achieved by accumulation of solutes which results in the maintenance of a positive turgor potential. In this study, the effect of water deficit on sugar uptake was investigated in leaf discs of Phaseolus coccinius L. (cv. Scarlet). Evidence is presented that cell turgor affects the kinetics of sugar transport at the membrane level. Uptake kinetics of sucrose, glucose and 3-O-methyl glucose by tissues equilibrated in solutions of relatively high (200–400 mOsm) osmotic concentration consisted of a sat-urable and a linear component. Low external osmotic concentration i.e., high cellular turgor inhibited the saturating component of sucrose uptake, resulting in a linear uptake profile. However, high cell turgor had no effect on glucose or 3-O-methyl glucose uptake kinetics. The effect of turgor versus osmotic component of water potential was differentiated by comparing responses to non-penetrating (manmtol) or polyethylene glycol, (3350) and penetrating (ethylene glycal) osmotica. Changes in sucrose uptake rates and kinetics were due to changes in cellular turgor and not osmotic potential. Furthermore, at low cellular turgor, a net increase in sucrose uptake occurred as a consequence of enhanced influx rates and not as a result of reduced efflux rates. The data are consistent with previous findings that sugar uptake rates are enhanced under low turgor. We present first evidence indicating that the mechanism by which higher rates of sucrose uptake are maintained underwater deficit conditions is by the activation of the saturable transport system. This mechanism supports previous suggestions that changes in cell turgor are sensed and manifested at the membrane level.     

Turgor and net ion flux responses to activation of the osmotic MAP kinase cascade by fludioxonil in the filamentous fungus Neurospora crassa

The internal hydrostatic pressure (turgor) of the filamentous fungus Neurospora crassa is regulated at about 400–500 kiloPascals, primarily by an osmotic MAP kinase cascade which activates ion uptake from the extracellular medium and glycerol synthesis. In the absence of hyperosmotic stress, the phenylpyrrole fungicide fludioxonil activates the osmotic MAP kinase cascade, resulting in cell death. Turgor, the electrical potential and net ion fluxes were measured after treatment with fludioxonil. In wildtype, fludioxonil causes a hyperpolarization of the plasma membrane and net H⁺ efflux from the cell, consistent with activation of the H⁺-ATPase. At the same time, net K⁺ uptake occurs, and turgor increases (about 2-fold above normal levels). None of these changes are observed in the os–2 mutant (which lacks a functional MAP kinase, the last of the three kinases in the osmotic MAP kinase cascade). Tip growth ceases as hyperpolarization, net ion flux changes, and turgor increases begin. The inappropriate turgor increase is the probable cause of eventual lysis and death. The results corroborate a multi-pathway response to hyperosmotic stress that includes activation of plasma membrane transport. The relation to cell expansion (tip growth) is not direct. Increases in turgor due to ion transport might be expected to increase growth rate, but this does not occur. Instead, there must be a complex regulatory interplay between the growth and the turgor driving force, possibly mediated by regulation of cell wall extensibility.     

Binding of SEC11 Indicates Its Role in SNARE Recycling after Vesicle Fusion and Identifies Two Pathways for Vesicular Traffic to the Plasma Membrane

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins drive vesicle fusion in all eukaryotes and contribute to homeostasis, pathogen defense, cell expansion, and growth in plants. Two homologous SNAREs, SYP121 (=SYR1/PEN1) and SYP122, dominate secretory traffic to the Arabidopsis thaliana plasma membrane. Although these proteins overlap functionally, differences between SYP121 and SYP122 have surfaced, suggesting that they mark two discrete pathways for vesicular traffic. The SNAREs share primary cognate partners, which has made separating their respective control mechanisms difficult. Here, we show that the regulatory protein SEC11 (=KEULE) binds selectively with SYP121 to affect secretory traffic mediated by this SNARE. SEC11 rescued traffic block by dominant-negative (inhibitory) fragments of both SNAREs, but only in plants expressing the native SYP121. Traffic and its rescue were sensitive to mutations affecting SEC11 interaction with the N terminus of SYP121. Furthermore, the domain of SEC11 that bound the SYP121 N terminus was itself able to block secretory traffic in the wild type and syp122 but not in syp121 mutant Arabidopsis. Thus, SEC11 binds and selectively regulates secretory traffic mediated by SYP121 and is important for recycling of the SNARE and its cognate partners.     

MsbA ATP-binding cassette (ABC) transporter of E. coli: structure and possible flippase mechanism

ATP-binding cassette (ABC) transporters utilize the energy present in cellular ATP to drive the translocation of structurally diverse set of solutes across the membrane barriers of eubacteria, archaebacteria and eukaryotes. In bacteria, these transporters are considered to be important virulence factors because they play role in nutrient uptake and in the secretion of toxins. The advances in structural determination and functional analysis of bacterial transporters have greatly increased our understanding of the mechanism of transport of these ABC transporters. Although progress in the field of structural biology has been made with the prokaryotic family members, it is likely that eukaryotic transporters will utilize the same mechanisms for translocation process. In this review, we summarize the function of the known MsbA ABC transporters in E. coli and mechanistic insights from structural and possible flippase mechanism studies.     

The Sec34/35 Golgi transport complex is related to the exocyst, defining a family of complexes involved in multiple steps of membrane traffic.

The specificity of intracellular vesicle transport is mediated in part by tethering factors that attach the vesicle to the destination organelle prior to fusion. We have identified a protein, Dor1p, that is involved in vesicle targeting to the yeast Golgi apparatus and found it to be associated with seven further proteins. Identification of these revealed that they include Sec34p and Sec35p, the two known components of the Sec34/35 complex previously proposed to tether vesicles to the Golgi. Of the six previously uncharacterized components, four have homologs in higher eukaryotes, including a subunit of a mammalian Golgi transport complex. Furthermore, several of the proteins show distant homology to components of two other putative tethering complexes, the exocyst and the Vps52/53/54 complex, revealing that tethering factors involved in different membrane traffic steps are structurally related.     

Probing intracellular vesicle trafficking and membrane remodelling by cryo-EM

Protein transport between the membranous compartments of the eukaryotic cells is mediated by the constant fission and fusion of the membrane-bounded vesicles from a donor to an acceptor membrane. While there are many membrane remodelling complexes in eukaryotes, COPII, COPI, and clathrin-coated vesicles are the three principal classes of coat protein complexes that participate in vesicle trafficking in the endocytic and secretory pathways. These vesicle-coat proteins perform two key functions: deforming lipid bilayers into vesicles and encasing selective cargoes. The three trafficking complexes share some commonalities in their structural features but differ in their coat structures, mechanisms of cargo sorting, vesicle formation, and scission. While the structures of many of the proteins involved in vesicle formation have been determined in isolation by X-ray crystallography, elucidating the proteins' structures together with the membrane is better suited for cryogenic electron microscopy (cryo-EM). In recent years, advances in cryo-EM have led to solving the structures and mechanisms of several vesicle trafficking complexes and associated proteins.     

Constant change: dynamic regulation of membrane transport by calcium signalling networks keeps plants in tune with their environment

Despite substantial variation and irregularities in their environment, plants must conform to spatiotemporal demands on the molecular composition of their cytosol. Cell membranes are the major interface between organisms and their environment and the basis for controlling the contents and intracellular organization of the cell. Membrane transport proteins (MTPs) govern the flow of molecules across membranes, and their activities are closely monitored and regulated by cell signalling networks. By continuously adjusting MTP activities, plants can mitigate the effects of environmental perturbations, but effective implementation of this strategy is reliant on precise coordination among transport systems that reside in distinct cell types and membranes. Here, we examine the role of calcium signalling in the coordination of membrane transport, with an emphasis on potassium transport. Potassium is an exceptionally abundant and mobile ion in plants, and plant potassium transport has been intensively studied for decades. Classic and recent studies have underscored the importance of calcium in plant environmental responses and membrane transport regulation. In reviewing recent advances in our understanding of the coding and decoding of calcium signals, we highlight established and emerging roles of calcium signalling in coordinating membrane transport among multiple subcellular locations and distinct transport systems in plants, drawing examples from the CBL‐CIPK signalling network. By synthesizing classical studies and recent findings, we aim to provide timely insights on the role of calcium signalling networks in the modulation of membrane transport and its importance in plant environmental responses.     

Alkali cation exchangers: roles in cellular homeostasis and stress tolerance

Uptake and translocation of cations play essential roles in plant nutrition, signal transduction, growth, and development. Among them, potassium (K+) and sodium (Na+) have been the focus of numerous physiological studies because K+ is an essential macronutrient and the most abundant inorganic cation in plant cells, whereas Na+ toxicity is a principal component of the deleterious effects associated with salinity stress. Although the homeostasis of these two ions was long surmised to be fine tuned and under complex regulation, the myriad of candidate membrane transporters mediating their uptake, intracellular distribution, and long-distance transport is nevertheless perplexing. Recent advances have shown that, in addition to their function in vacuolar accumulation of Na+, proteins of the NHX family are endosomal transporters that also play critical roles in K+ homeostasis, luminal pH control, and vesicle trafficking. The plasma membrane SOS1 protein from Arabidopsis thaliana, a highly specific Na+/H+ exchanger that catalyses Na+ efflux and that regulates its root/shoot distribution, has also revealed surprising interactions with K+ uptake mechanisms by roots. Finally, the function of individual members of the large CHX family remains largely unknown but two CHX isoforms, AtCHX17 and AtCH23, have been shown to affect K+ homeostasis and the control of chloroplast pH, respectively. Recent advances on the understanding of the physiological processes that are governed by these three families of cation exchangers are reviewed and discussed.