Isolation of native plasma membrane H⁺-ATPase (Pma1p) in both the active and basal activation states

The yeast plasma membrane H(+)-ATPase Pma1p is a P-type ATPase that energizes the yeast plasma membrane. Pma1p exists in two activation states: an autoinhibited basal state and an activated state. Here we show that functional and stable Pma1p can be purified in native form and reconstituted in artificial liposomes without altering its activation state. Acetylated tubulin has previously been reported to maintain Pma1p in the basal state but, as this protein was absent from the purified preparations, it cannot be an essential component of the autoinhibitory mechanism. Purification of and reconstitution of native Pma1p in both activation states opens up for a direct comparison of the transport properties of these states, which allowed us to confirm that the basal state has a low coupling ratio between ATP hydrolysis and protons pumped, whereas the activated state has a high coupling ratio. The ability to prepare native Pma1p in both activation states will facilitate further structural and biochemical studies examining the mechanism by which plasma membrane H(+)-ATPases are autoinhibited

Membrane transport, sensing and signaling in plant adaptation to environmental stress

Plants are generally well adapted to a wide range of environmental conditions. Even though they have notably prospered in our planet, stressful conditions such as salinity, drought and cold or heat, which are increasingly being observed worldwide in the context of the ongoing climate changes, limit their growth and productivity. Behind the remarkable ability of plants to cope with these stresses and still thrive, sophisticated and efficient mechanisms to re-establish and maintain ion and cellular homeostasis are involved. Among the plant arsenal to keep homeostasis are efficient stress sensing and signaling mechanisms, plant cell detoxification systems, compatible solute and osmoprotectant accumulation and a vital rearrangement of solute transport and compartmentation. The key role of solute transport systems and signaling proteins in cellular homeostasis is addressed in the present work. The full understanding of the plant cell complex defense mechanisms under stress may allow for the engineering of more tolerant plants or the optimization of cultivation practices to improve yield and productivity, which is crucial in the present time as food resources are progressively scarce.This work was supported by the Portuguese Foundation for Science and Technology (FCT) (research project ref. PTDC/AGR-ALI/100636/2008; to A. Conde, grant ref. SFRH/BD/47699/2008)

Light-Induced Stomatal Opening Is Affected by the Guard Cell Protein Kinase APK1b.

Guard cells allow land plants to survive under restricted or fluctuating water availability. They control the exchange of gases between the external environment and the interior of the plant by regulating the aperture of stomatal pores in response to environmental stimuli such as light intensity, and are important regulators of plant productivity. Their turgor driven movements are under the control of a signalling network that is not yet fully characterised. A reporter gene fusion confirmed that the Arabidopsis APK1b protein kinase gene is predominantly expressed in guard cells. Infrared gas analysis and stomatal aperture measurements indicated that plants lacking APK1b are impaired in their ability to open their stomata on exposure to light, but retain the ability to adjust their stomatal apertures in response to darkness, abscisic acid or lack of carbon dioxide. Stomatal opening was not specifically impaired in response to either red or blue light as both of these stimuli caused some increase in stomatal conductance. Consistent with the reduction in maximum stomatal conductance, the relative water content of plants lacking APK1b was significantly increased under both well-watered and drought conditions. We conclude that APK1b is required for full stomatal opening in the light but is not required for stomatal closure

The Plasma Membrane H+-ATPase - Identification of a 14-3-3 binding motif

The P-type plasma membrane H+-ATPases form a group of proteins only found in plants and fungi. The pumping of protons across the plasma membrane, energized by ATP hydrolysis, creates an electrochemical gradient that is essential for solute transport and internal pH regulation. The H+-ATPase genes are present as multigene families in the genomes of higher plants and all cell types investigated express some H+-ATPase gene. The fundamental importance of the electrochemical gradient makes precise regulation of the H+-ATPase important. Several internal and external factors, such as hormones, light, pH, and fungal toxins, are involved in regulating the plant plasma membrane H+-ATPase activity. Besides the direct regulation by pH and ATP availability, the activity is controlled by an autoinhibitory C-terminal domain in the H+-ATPase. Removing this C-terminal domain by proteolysis or by fusicoccin-induced 14-3-3 binding irreversibly activates the enzyme. Normally, 14-3-3 binds to phosphorylated motifs and by incubating spinach leaves with 32P-orthophosphate and the fungal toxin fusicoccin in vivo it was possible to radiolabel the H+-ATPase. The radiolabeling could be removed by proteolysis and sequencing the released radiolabeled peptides identified the phosphorylated amino acid as the penultimate threonine in the C terminus, in the relatively conserved motif QQXYTV. This phosphorylated threonine is essential for 14-3-3 binding in the absence of fusicoccin, whereas fusicoccin-induced 14-3-3 binding occurs regardless of phosphorylation but still requires the YTV residues. The physiological importance of this motif was shown by heterologous expression of a plant H+-ATPase in yeast. Mutations in the motif abolished or heavily reduced 14-3-3 binding and activation of the plant H+-ATPase. In vitro phosphorylation of isolated plasma membranes with [ g -32P]ATP radiolabels the H+-ATPase in a calcium-dependent way and creates a 14-3-3 binding site in the H+-ATPase, containing a phosphothreonine. The H+-ATPase isoforms AHA1 and AHA2 are present in Arabidopsis leaf plasma membranes under normal conditions; fusicoccin treatment induces expression of three additional isoforms, AHA3, AHA8, and AHA11. Five 14-3-3 isoforms, epsilon, mu, nu, omega, and upsilon, are associated with the plasma membrane, where the H+-ATPase is the main target for 14-3-3 binding; after infiltration with fusicoccin there is a change in isoforms, omega disappears and the chi isoform appears. In summary, the data show that in vivo phosphorylation of the penultimate threonine in the motif QQXYTV regulates the H+-ATPase activity by 14-3-3 binding and that a calcium-dependent protein kinase activity phosphorylating this threonine in vitro is present in the plasma membrane. The appearance of additional H+-ATPase isoforms and the shift in 14-3-3 isoforms after fusicoccin-treatment is interesting and further research might answer questions regarding 14-3-3 isoform specificity and the function of the different H+-ATPase isoforms, in e.g. adaptation to stress

Tillämpning av DMAIC-metodiken i tjänsteföretag : en fallstudie genomförd på EuroMaint Rail AB

Validerat; 20101217 (root