Characterization of Tonoplast Polypeptides Isolated from Corn Seedling Roots

Abstract

Tonoplast vesicles were isolated by discontinuous sucrose gradient centrifugation in the presence of Mg2+ from 5 day old corn (Zea mays L., Golden Cross Bantam) seedling roots. Marker enzyme assays indicated only a low degree of cross-contamination of tonoplast vesicles at the 10/23% (weight/weight) interface by other membrane components. Severalfold enrichment of tonoplast ATPase and pyrophosphatase was indicated in tonoplast fractions by dot blot studies with antibodies against an oat tonoplast ATPase and a mung bean tonoplast pyrophosphatase. Comparison of two-dimensional electrophoretic gels of tonoplast and microsomal membrane polypeptides revealed approximately 68 polypeptides to be specific to tonoplast by silver staining. Immunoblot analysis with antibodies against a tonoplast holoenzyme ATPase from oat roots revealed the presence of the 72, 60, and 41 kilodalton polypeptides in isolated tonoplast vesicles from corn roots. Affinity blotting with concanavalin A and secondary antibodies indicated the degree of glycosylation of tonoplast polypeptides, where 21 of 68 tonoplast-specific polypeptides contained detectable carbohydrate moieties. Salt and NaOH washes removed 38 of the tonoplast-specific polypeptides, indicating a peripheral association with the membrane. Thirteen of the peripheral polypeptides and eight of the integral polypeptides were identified as glycoproteins. This information on the polypeptide composition of the tonoplast of root cells will aid in gaining insight into the role of this membrane in controlling vacuolar functions.The central vacuole of mature plant cells usually occupies 80% or more of the intracellular volume. It is now recognized that vacuoles function in osmotic-based tissue support, ion balance and storage, metabolite storage and sequestration, and senescence and lysosomal compartmentation (32). Many of these functions are mediated by the tonoplast membrane, which separates the neutral cytosol from the acidic and hydrolytic vacuolar sap. Since the development of techniques for the routine isolation of vacuoles, much information has been obtained on the properties of the tonoplast and the composition of the vacuolar sap (3). It is recognized that a Mg+-ATPase or pyrophosphatase generates a proton motive force across the tonoplast that can be utilized to energize the transport of various solutes. Evidence for Ca2+/H', Na+/H+, sucrose/H+ antiport, and NO3-/H' symport in the tonoplast has been provided (13). However, apart from the isolation of proton-translocating ATPase and pyrophosphatase, very little is known about tonoplast membrane proteins that conduct the rapid and specific exchange of ions, metabolites, and proteins between the cytosol and vacuolar sap.Characterization of the protein composition of the tonoplast is an important initial step in understanding the mechanism by which the vacuole functions as an active metabolic compartment within the plant cell. Customarily, a particular membrane is identified and contamination assessed by the distribution of a s...

Studies on vacuole regeneration in evacuolated tobacco mesophyll protoplasts. Protein analysis by one‐ and two‐dimensional microgel‐electrophoresis

“…When the same sera were used on total protein extracts from a heterotrophically grown suspension culture, the additional bands did not appear, whereas the subunits of the vacuolar ATPase were detected equally well (not shown). The additional reactions discus.sed here have not been described by other authors (e,g, DuPont et al, 1988, Glenn et al, 1989, Ni and Beevers 1991. Since they used etiolated plants or chlorophyll-free tissues, this further supports our hypothesis.…”

“…In particular, the ATPase is a protein well conserved in evolution Nelson 1989, Nelson andTaiz 1989). Therefore, crossreactions of antibodies directed against this protein occur even between distantly related species (e,g, DuPont et al 1988, Ni andBeevers 1991). The anti-sera used here were raised against Kalanchoe ATPase, and detected tbe homologous proteins in tobacco extracts.…”