Planta (1996)198:95-103 P l a n t ~ 9 Springer-Vedag 1996 Immunological detection of tonoplast polypeptides in the plasma membrane of pea cotyledons David G. Robinson 1, Hans-Peter Haschke z, Giselbert Hinz 1, Birgit Hoh ~, Masayoshi Maeshima 3, Francis Marty 4 1 Pflanzenphysiologisches Institut, Universit~it Grttingen, Untere Karspiile 2, D-37073 Grttingen, Germany 2 Institut fiir Pflanzenphysiologie und Mikrobiologie, Freie Universit~it Berlin, Krnigin-Luise-Strasse 12-16a, D-14195 Berlin, Germany 3 Laboratory of Biochemistry,School of Agricultural Sciences,Nagoya University, Japan '* Laboratoire de Phytobiologie Cellulaire, Universit6 de Bourgogne, Dijon, France Received: 14 March 1995/Accepted: 12 April 1995 Abstract. The tonoplast is usually characterized by the presence of two electrogenic proton pumps: a vacuolar- type H+-ATPase and a pyrophosphatase, as well as a putative water-channel-forming protein (y-TIP). Using a post-embedding immunogold labelling technique, we have detected the presence of these transport-protein complexes not only in the tonoplast, but also in the plasma membrane and trans Golgi elements of maturing pea (Pisum sativum L.) cotyledons. These ultrastructural observations are supported by Western blotting with highly purified plasma-membrane fractions. In contrast to the vacuolar-type H+-ATPase, whose activity was not measurable, considerable pyrophosphatase activity was detected in the plasma-membrane fraction. These results are discussed in terms of a possible temporary repository for tonoplast proteins en route to the vacuole. Key words: V-ATPase-immunocytochemistry - Pea coty- ledon- Plasma membrane - Putative y-TIP - Pyrophos- phatase - Tonoplast markers Introduction The membrane of the plant vacuole, the tonoplast (TP), is characterized by three protein complexes (see Taiz 1992, for a review). Two of them represent electrogenic pumps: an H+-ATPase of the vacuolar-type (V-ATPase; Ward and Sze 1992), similar to that described for endomem- branes in animal cells (Nelson 1992), and an H+-pyro - phosphatase (PPase, Rea and Poole 1993). The third, which in amount is more abundant than the other two, has a molecular mass of around 25 kDa and has been termed tonoplast intrinsic-protein (TIP, Johnson et al. Abbreviations: 1327 = 27-kDa tonoplast intrinsic protein, a putative y-TIP; PM = plasma membrane; PPase = pyrophosphatase; TIP = tonoplast intrinsic protein; TP = tonoplast; V(P)-ATPase = vacuolar (plasma membrane)-typeproton-transporting ATPase Correspondence to: D.G. Robinson; FAX: 49 (551) 397833 1990). There are two major isoforms of TIP in higher plants: y-TIP, which is present in the TP of vegetative organs (Marty-Mazars et al. 1995), and an a-TIP, which is expressed in seed tissue during later stages of development (H/Sfte et al. 1992). Recent research strongly suggests that y-TIP functions as a water channel (Maurel et al. 1993), and that it is related to similar channel proteins in bacteria and animals, collectively termed the MIP-family (major intrinsic protein, Pao et al. 1991). Normally V-ATPases are regarded as being absent from the plasma membrane (PM) but, in the animal king- dom, there are some notable exceptions, all of which represent cell types specialized for acid secretion. These include neutrophils and macrophages (e.g. Swallow et al. 1993), where the H+-ATPases serve in cytoplasmic pH regulation and in the activation of extracellular hydro- lases; osteoclasts (e.g. Chatterjee et al. 1992), which partici- pate in acid-mediated bone resorption; intrahepatic bile- duct epithelial cells which secrete HCO3 into bile and H § to periductuolar fluid (Villanger et al. 1993); epithelial cells in the kidney (e.g. Gluck et al. 1992) and bladder (e.g. Brown et al. 1987) of mammals; and gastrointestinal epi- thelia of insects (e.g. Klein 1992). In addition, it has re- cently been shown that the high cytoplasmic pH in human tumorigenic cell lines is a result of a missorting of intracel- lular vesicle traffic leading to an overexpression of V- ATPases in the PM (Martinez-Zaguilan et al. 1993). There is thus no a priori reason for assuming that V-ATPases should be restricted to the TP of plant cells, especially since the cell wall is also an acid milieu. The literature does in fact contain reports of both V-ATPase activities (Bush 1989; Williams et al. 1990) and V-ATPase-like polypept- ides (Kimura et al. 1988; Mito et al. 1988) in plant PM prepared by phase partitioning. Similarly, PPase activity has also been measured in PM-enriched fractions from a variety of plant tissues (Petel and Gendraud 1989; Macri and Vianello 1990; Williams et al. 1990; Faraday and Spanswick 1992). However, in all of these cases the possi- bility that this merely reflects TP contamination cannot be excluded. The same line of argument might also apply to y-TIP since its animal homologues, in particular CHIP-28