1. Regulation of the plasma membrane H-ATPase activity

The plasma membrane forms a barrier between the cell and its surroundings. This position as the outer permeability barrier gives the plasma membrane an unique role in controlling cell life. Important functions ascribed to the plasma membrane are i.e. balanced transport of specific molecules (ions, solutes, photosynthetic products etc) into or out of the cell to provide a relatively constant intracellular milieu, environmental signal recognition, and transduction of these signals into intracellular responses, and to provide structural links between the cytoskeleton and extracellular components to influence cell flexibility. The plasma membrane proton pumping ATPase (H-ATPase) has a key role in the regulation of many processes in the plant. It creates the electrochemical gradient (proton gradient and membrane potential) across the plasma membrane that drives e.g. nutrient uptake in the roots and phloem loading in the sieve tubes, thereby regulating plant growth. Transient changes in the activity of the H-ATPase causes transient changes in the membrane potential leading to activation of, e.g., voltage-gated Ca² channels and thus Ca²-dependent signal transduction chains. Through enhanced proton extrusion into the apoplast the enzyme induces cell growth, and by controlling the pH in the cytoplasm it is believed to control cell division. The H-ATPase activity, in turn, is regulated by a number of factors including plant hormones, light, salinity, elicitors, wounding, and fungal toxins. Thus, the plasma membrane H-ATPase is likely to constitute a cross-point for several signal transduction pathways.

 

EARLIER RESULTS
The first clue as to how the H-ATPase can be regulated at the molecular level came when we in identified an autoinhibitory domain in the C-terminal region. Activation of proton pumping by the fungal toxin fusicoccin proceeds via this autoinhibitory domain and thus established a physiological role for this type of regulation. Activation of the H-ATPase is achieved through binding of 14-3-3 protein to the C-terminal region, an activation that is stabilized by fusicoccin. 14-3-3s usually bind to proteins having zinc-finger motifs or certain phosphorylated motifs.

A unique  phosphorylated 14-3-3 binding motif was identified in the C terminus of the H-ATPase. This motif, YpTV, where the phosphorylated Thr-948 represents the penultimate amino acid in the C terminus, is conserved and present in all Arabidopsis H-ATPase isoforms except one. The physiological importance of this motif was shown by heterologous expression of a plant H-ATPase in yeast. Mutations in the motif (replacing Thr-948 with Ala) could not complement the yeast H-ATPase (which the normal plant enzyme can) and abolished 14-3-3 binding and activation of the H-ATPase. Thus all data point to 14-3-3 being a natural  ligand of the H-ATPase.

Using surface plasmon resonance (Biacore) we showed that 14-3-3 isoforms differ in their affinity for this unique motif. To date, the generally held view is that there is no isoform specificity for target proteins containing the more common motifs, although exceptions are known. We identified five new 14-3-3 genes and showed expression of two of these, thus fifteen 14-3-3 genes are present in Arabidopsis. The Arabidopsis 14-3-3 gene map demonstrates a division of its 14-3-3s into two different groups in agreement with the two major branches observed in the phylogenetic tree.

FUTURE PLANS
Our aim is to resolve the detailed molecular mechanism behind regulation of the H-ATPase involving the autoinhibitory C-terminal domain. We aim to identify components involved, resolve their interactions at the molecular level, and elucidate the physiological role of this domain in the response to environmental factors. One of our goals is to identify the protein kinase(s)/phosphatase(s) phosphorylating/dephosphorylating the H-ATPase. We will investigate the regulatory properties of these enzymes with respect to their substrate (H-ATPase) and 14-3-3. The existence of fifteen 14-3-3 isoforms (13 expressed) and twelve H-ATPase genes (11 expressed) in Arabidopsis raises the question of whether isoform specificity exist, that is, does a certain isoform interact with a specific 14-3-3. Our surface plasmon resonance data (Biacore) indicates that some specificity may indeed exist. We want to investigate this in more detail We are using a number of up-to-date biochemical, molecular biology, and biophysical techniques to resolve these issues.

 

 

Other projects:

 

2. Plant signalling involving phospholipid-derived compounds

One of the most significant advances in animal cell biology was the recognition that phospholipids not only are structural components of membranes but also functions as sources of intracellular messengers. In particular, inositol phospholipids play multiple roles in cell signalling. They provide the cell with an astonishing variety of signals whose function we are only just beginning to understand in detail. The most well-known phosphoinositide signalling pathway (PI cycle) involves an agonist-induced hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP₂) by a specific phospholipase C (PI-PLC) to generate two intracellular messengers, diacylglycerol and inositol trisphosphate. The latter releases Ca² from intracellular stores which results in further specific downstream effects. PIP₂ is generated from phosphatidyl-inositol (PI) by phosphorylation first to phosphatidylinositol 4-phosphate (PI-4P or PIP) and then further to PIP₂. These reactions are catalyzed by PI and PIP kinases, respectively. Evidences are now accumulating that point in the direction for a role of the plant PI system in mediating transduction of environmental stress signals like osmotic stress, acid stress, light exposure, and pathogen attacks, as well as in pollen tube growth, nuclear function, and in regulating turgor changes which underlie stomatal opening and closure and the movements of leaves and flower parts. It is also becoming obvious that phosphoinositides (including those phosphorylated at the D3 position of the inositol ring) play a much broader and complex role in cell regulation than previously expected, e.g. as direct modulators of specific proteins like protein kinases, phospholipase D, diacylglycerol kinase, and some P-type ATPases, and in regulation of cytoskeletal dynamics and vesicular/membrane trafficking.

 

We are studying the regulation and localization of PI cycle enzymes in Arabidopsis and hope to advance the understanding of their role in signalling. We will in particular study the three enzymes (including some of their isoforms), PI-PLC, PIP kinase, and PI-kinase with respect to regulation by phosphorylation/ dephosphorylation, to reveal the nature of their membrane-associations and identify potential cellular components that may interact with specific domains in these enzymes under in vivo conditions. One interesting question is what the precise functions of the plant PI kinases are, i.e. the PI-4P generated. Given the higher abundance of PI 4-P (up to 35-fold) to PIP₂ in plant cells compared to animal cells PI 4-P may play a significant role in processes other than the classical signal transduction pathway.

 

3. Role of Ca²-binding proteins in Ca² homeostasis

The sensitivity and responses of the cell to various environmental stresses, such as cold, Ca² deficiency, and salinity, is dependent on its ability to sequester and use Ca² from internal stores. The ability to modulate intracellular Ca²pools could thus provide a means for plants to gain resistance to encountered external stresses. We focus on the endoplasmic reticulum (ER) localized Ca²-binding protein calreticulin (CRT), ER Ca²-ATPases, and the vacuolar Ca²/H antiports, with the aim to understand their detailed regulation and their role in maintaining and restoring ionic homeostasis in response to environmental factors i.e., their role in signal transduction.

 

Larsson, C., Sommarin, M., Widell, S. (). Isolation of highly purified plant plasma membranes and the separation of inside-out and right-side-out vesicles.  Methods Enzymol 228: 451-469

 

Svennelid, F., Olsson, A., Piotrowski, M., Rosenquist, M., Ottman, C., Larsson, C., Oecking, C., Sommarin, M. (). Phosphorylation of Thr-948 at the C terminus of the plasma membrane H-ATPase creates a binding site for the regulatory 14-3-3 protein. Plant Cell 11:

 

Rosenquist, M., Sehnke, P., Ferl, R.J., Sommarin, M., Larsson, C. (). Evolution of the 14-3-3 protein family: Does the large number of isoforms in multicellular organisms reflect functional specificity? J Mol Evol 51: 446-458

 

Rosenquist, M., Alsterfjord, M., Larsson, C., Sommarin, M. (). Data-mining the Arabidopsis genome reveals fifteen 14-3-3 genes. Expression demonstrated for two out of five novel genes. Plant Physiol 127: 142-149

 

Sehnke, P.C., Rosenquist, M., Alsterfjord, M., DeLille, J., Sommarin, M., Larsson, C., Ferl, R.J. () Evolution and isoform specificity of plant 14-3-3 proteins. Plant Mol Biol 50:

 

Pical, C., Westergren, T., Dove, S.K., Larsson, C., Sommarin, M. (). Salinity and hyperosmotic stress induce rapid increases in phosphatidylinositol 4, 5-bisphosphate, diacylglycerol pyrophosphate, and phosphatidylcholine in Arabidopsis thaliana cells. J  Biol Chem 274:

 

Otterhag, L., Sommarin, M., Pical, C. (). N-terminal EF-hand-like domain is required for phosphoinositide-specific phospholipase C activity in Arabidopsis thaliana. FEBS Lett 497: 165-170

 

Westergren, T., Sommarin, M., Pical, C. (). The Arabidopsis thaliana phosphatidylinositol phosphate kinase AtPIP5K1 synthesizes PtdIns(3,4)P₂ and PtdIns(4,5) P₂ in vitro, and is inhibited by phosphorylation. Biochem J 359: 583-589

 

Persson, S., Rosenquist, M., Sommarin, M. (). Identification of a novel calreticulin isoform (Crt2) in human and mouse. Gene 297: 151-158

 

Persson, S., Rosenquist, M., Svensson, K., Galvao, R., Boss, W.F., Sommarin, M. (). Phylogenetic analyses and expression studies reveal two distinct groups of calreticulin isoforms in higher plants. Plant Physiol 133: