Auxin Regulation of a Proton Translocating ATPase in Pea Root Plasma Membrane Vesicles

Abstract

ABSTRACrPea root microsomal vesicles have been fractionated on a Dextran step gradient to give three fractions, each of which carries out ATP-dependent proton accumulation as measured by fluorescence quenching of quinacrine. The fraction at the 4/6% Dextran interface is enriched in plasma membrane, as determined by UDPG sterol glucosyltransferase and vanadate-inhibited ATPase. The vanadate-sensitive phosphohydrolase is not specific for ATP, has a K, of about 0.23 millimolar for MgATP, is only slightly affected by K' or Cl-and is insensitive to auxin. Proton transport, on the other hand, is more specific for ATP, enhanced by anions (NO3-> Cl-) and has a K. of about 0.7 millimolar. Auxins decrease the K., to about 0.35 millimolar, with no significant effect on the V,,,., while antiauxins or weak acids have no such effect. It appears that auxin has the ability to alter the efficiency of the ATP-driven proton transport.The plasma membrane of plant cells contains an ATPase which is responsible for the electrogenic export of protons to the apoplastic solution (26). Its activity can be studied with isolated, inside-out plasma membrane vesicles, since they can retain the ability to transport protons in response to MgATP (25,27,29 on wet filter paper. After 3 d, the apical (2 cm) tips of the roots were harvested.Isolation of Sealed Microsomal Vesicles. All procedures were carried out at 4°C. The roots were first chopped with a razor blade and then homogenized using a mortar and pestle in 1 ml/ g fresh weight of buffer 1 (6% [w/w] sorbitol, 3 mm EDTA, 0.1 % BSA, 25 mM Tris, 20 mM mercaptoethanol, pH 7.0, with Mes). The homogenate was strained through four layers ofcheesecloth, the residue was rehomogenized in 1 ml/g fresh weight of buffer I and refiltered. The total filtrate was centrifuged at 6,000g for 20 min. The supematant was then centrifuged at 1 l0,OOOg for 30 min (Rotor type 30, Beckman L5-50 centrifuge) to obtain the microsomal pellet. The pellet was resuspended in 5 ml of buffer I and layered over a discontinuous Dextran T70 gradient consisting of 7 ml of 10%, 7 ml of 6%, and 7 ml of 4% Dextran T70 (w/w) in buffer I. This was centrifuged at 70,000g for 2 h (Rotor SW 27). The membrane bands at the interfaces were removed with a Pasteur pipette (fraction A, 0/4% interface; B, 4/6%; C, 6/10%) and diluted with buffer 11 (4.5% sorbitol, 25 mm Tris, pH 7.0, with Mes). After centrifugation at 1 l0,OOOg for 30 min, each pellet was resuspended in buffer II (except where indicated) and was used immediately for enzyme and proton translocation experiments.Enzyme Assays. ATPase activity was assayed in 0.5 ml containing 25 mM Tris-Mes pH 7.0, 10 mm MgSO4, 0.1 mM ammonium molybdate, 1 mM NaN3, 0.067 to 2.5 mm NaATP, and salts as indicated. The presence of sodium ions in NaATP did not affect the results, as Tris-ATP gave identical results. Reactions were initiated by addition of membrane protein (10-50 jig). After 10 to 30 min at 30°C, the reaction was stopped with 1 ml ice-cold 0.5% ammonium molybdate + 0.1% SDS in 0.72 N H2SO...

ADP‐ or pyrophosphate‐dependent proton pumping of pea stem tonoplast‐enriched vesicles

“…This H+-ADP/PPiase, on the other hand, appears to be different, in its characteristics, from the nitrate-inhibited, chloride-stimulated and vanadate-insensitive H+-ATPase of pea stem microsomes [15,16] and tonoplast-enriched fractions (results to be published), and from the vanadate-inhibited, nitrate-insensitive H÷-ATPase of pea root plasmalemma-enriched fraction [19]. The ATP-and ADP-or PPi-dependent proton pumping activity seems to be located on the same type of vesicles, since the ATP-generated zapH appears to counteract the ADP-or PPi-dependent proton pumping activity ( fig.lE,F).…”

“…ATPase has a proton translocating activity [15,16] linked to the generation of an electrochemical potential [17] and to a H÷/Ca 2+ antiport [18]. In addition, a H÷-ATPase has also been shown in pea root plasma membrane-enriched vesicles [19].…”

Latency of Plasma Membrane H+-ATPase in Vesicles Isolated by Aqueous Phase Partitioning

“…ATPase action has frequently been studied using vesicles prepared by sucrose or dextran density-gradient centrifugation (5,7,27,28). This method yields sealed, inside-out PM vesicles in which access of ATP to the ATPase is not a problem, and in…”

Auxin-binding protein located in the endoplasmic reticulum of maize shoots: molecular cloning and complete primary structure.

“…Rapid responses to auxin have been observed in organelles such as plasma membrane (3)(4)(5)(6), Golgi apparatus (7), endoplasmic reticulum (ER) (8), and nucleus (2,9,10) as well as in the cell wall (11). The specificity of auxin is supposed to be due to the presence of a specific receptor(s) (12).…”