Proton Transport and Phosphorylation of Tonoplast Polypeptides from Zucchini Are Stimulated by the Phospholipid Platelet-Activating Factor

Abstract

The ether phospholipid platelet-activating factor and certain similar phospholipids, including lysophosphatidylcholine, are known to stimulate both H' transport and protein phosphorylation in plant microsomal membranes. In the present work, several polypeptides in highly purified tonoplast membranes from zucchini (Cucurbita pepo L.) showed platelet-activating factor-dependent phosphorylation. Comparison of protein phosphorylation in different membrane fractions separated by sucrose step density gradient centrifugation indicated that some of the phosphoproteins were contaminants or were common to several membrane fractions, but platelet-activating factor-dependent phosphorylation of peptides at 30, 53, and perhaps 100 kilodaltons was tonoplast specific. The phosphoprotein of 53 kilodaltons was shown by three different approaches (one-and two-dimensional polyacrylamide gel electrophoresis, western blots, and immunoprecipitation) to cross-react with antibody raised against the B subunit of the tonoplast ATPase from red beet (Beta vulgaris L.).Although the vacuolar H+-ATPases of plants, animals, and fungi have been receiving increasing attention, much of the current work is focused on their structure and evolution (9,14,18) and relatively little is known of their regulation. Increased activity of the enzyme has been observed as an adaptation to salt stress (19) and during adaptation to CAM (27), and regulation is also assumed to occur in the turgor responses of guard cells and other systems.In vitro, proton transport and ATPase activity in a light microsomal fraction from zucchini (Cucurbita pepo L.) were shown to be increased in the presence of the ether phospholipid 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine (12,20,24 enhanced influx of calcium ions into platelets and the tumover of phosphorylated phosphatidylinositols (5). In plants, PAF presumably mimicks the action of PAF-like plant phospholipids (12, 24) such as lysophosphatidylcholine. A protein kinase stimulated by PAF, lysophospholipids, and Ca2" has been demonstrated in higher plants (12,21,23,24). We proposed that the PAF/lysophospholipid-stimulated protein kinase could be involved in a signal transduction chain participating in cell regulation in higher plants (20)(21)(22)(23)(24).Membrane fractionation studies showed that H+ transport in zucchini microsomes was attributable mostly to the tonoplast (12). The present work, therefore, examines in more detail the phosphorylation of tonoplast proteins and its relationship to the activation of tonoplast H+ transport by PAF. MATERIALS AND METHODS Plant MaterialZucchini seeds (Cucurbita pepo L.) were surface-sterilized with sodium hypochlorite and grown for 4 d in the dark at 280C on vermiculite. Hypocotyl hooks of about 1 cm were used for membrane preparations. Membrane PreparationsThe preparation of light microsomes from zucchini has been described (20). Chopped hypocotyls were degassed in homogenization buffer (1 g:1 mL) containing 4% (v/v) ethanolamine, 0.4 M sodium ,B-glycerophosphate, 20 mm...

Resolution of Subunit Interactions and Cytoplasmic Subcomplexes of the Yeast Vacuolar Proton-translocating ATPase

“…We believe that these are due to modifications of Vma2p which alter its charge-to-mass ratio. It has been shown that Vma2p is phosphorylated in both plant and animal systems (47,48), and there is recent evidence that it is also phosphorylated in yeast. 2 At least one modification may be dependent upon Vma4p, since an unusual band appears in gel-resolved extracts from the ⌬vma4 strain, with Vma2p as the only detectable component.…”

SOS2 Promotes Salt Tolerance in Part by Interacting with the Vacuolar H+-ATPase and Upregulating Its Transport Activity

“…NTAP-SOS2 purification identified five subunits (A, B1/2/3, C, E1, and G1) of the V-ATPase peripheral (cytoplasmic) sector as being components of a SOS2-containing protein complex. Of these subunits, VHA-A is responsible for the binding and hydrolysis of ATP, VHA-B is involved in noncatalytic ATP binding and, it has also been suggested, has a regulatory role, and VHA-C, VHA-E, and VHA-G are involved in the assembly and coupling of the peripheral and integral membrane sectors of the V-ATPase complex (29,34,44,51,54).…”

“…Our previous yeast two-hybrid screen using SOS2 as the bait protein identified not only ABI2 (33) but also VHA-B and several other clones as putative interactors with SOS2 (data not shown). VHA-B, together with VHA-A, controls the binding of ATP, whose hydrolysis is then catalyzed by VHA-A alone while VHA-B, it has been suggested, has a role in regulating the activity of the V-ATPase complex (29,44,54). To confirm the interaction between VHA-B proteins and SOS2, the entire open reading frames of VHA-B1 and VHA-B2 were fused with the GAL4 activation domain in the prey plasmid pACT2 and cotransformed with the bait plasmid pAS-SOS2, pAS-SOS2 K40N, or pAS-SOS1 (33) into the yeast strain Y190.…”

Gibberellic Acid Induces Vacuolar Acidification in Barley Aleurone.

“…Little is known about the mechanisms that regulate V-ATPase and V-PPase activities in plants. It has been postulated that phosphorylation of the B subunit of the V1 domain of the V-ATPase plays a role in regulating the activity of the enzyme (Martiny-Baron et al, 1992). Other mechanisms proposed to regulate the V-ATPase are cytosolic pH (Davies et al, 1994) and the redox state of the cytosol (Feng and Forgac, 1992;Muller et al, 1996).…”