Enhanced separation of membranes during free flow zonal electrophoresis in plants.
Free flow zonal electrophoresis (FFZE) is a versatile technique that allows for the separation of cells, organelles, membranes, and proteins based on net surface charge during laminar flow through a thin aqueous layer. We have been optimizing the FFZE technique to enhance separation of plant vacuolar membranes (tonoplast) from other endomembranes to pursue a directed proteomics approach to identify novel tonoplast transporters. Addition of ATP to a mixture of endomembranes selectively enhanced electrophoretic mobility of acidic vesicular compartments during FFZE toward the positive electrode. This has been attributed to activation of the V-ATPase generating a more negative membrane potential outside the vesicles, resulting in enhanced migration of acidic vesicles, including tonoplast, to the anode (Morré, D. J.; Lawrence, J.; Safranski, K.; Hammond, T.; Morré, D. M. J. Chromatogr., A 1994, 668, 201-213). We confirm that ATP does induce a redistribution of membranes during FFZE of microsomal membranes isolated from several plant species, including Arabidopsis thaliana, Thellungiella halophila, Mesembryanthemum crystallinum, and Ananas comosus. However, we demonstrate, using V-ATPase-specific inhibitors, nonhydrolyzable ATP analogs, and ionophores to dissipate membrane potential, that the ATP-dependent migrational shift of membranes under FFZE is not due to activation of the V-ATPase. Addition of EDTA to chelate Mg2+, leading to the production of the tetravalent anionic form of ATP, resulted in a further enhancement of membrane migration toward the anode, and manipulation of cell surface charge by addition of polycations also influenced the ATP-dependent migration of membranes. We propose that ATP enhances the mobility of endomembranes by screening positive surface charges on the membrane surface.
Barkla
Bronwyn J
BJ
Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3, Colonia Miraval, Cuernavaca, Morelos, México. bronwyn@ibt.unam.mx
Vera-Estrella
Rosario
R
Pantoja
Omar
O
D000255:Adenosine Triphosphate / Q000737:chemistry*; D002458:Cell Fractionation / Q000379:methods*; D004586:Electrophoresis / Q000379:methods*; D007425:Intracellular Membranes / Q000737:chemistry*; D059828:Plant Cells; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D010944:Plants / Q000737:chemistry* / Q000648:ultrastructure; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry; D014617:Vacuoles / Q000737:chemistry*
D010940:Plant Proteins; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases