ABSTRACEOsmotin is a cationic protein with a pl2 value >8.2 and accounts for 10 to 12% of total cellular protein (as determined by spectrophotometric measurement of Coomassie stained gels) in NaCl and polyethylene glycol adapted tobacco cells (23). In unstressed unadapted cells, an immunologically related 26 kD protein with a lower pl is also synthesized but is not accumulated under normal growth conditions (23). Osmotin synthesis is regulated by abscisic acid, but its accumulation depends on adjustment of the cells to NaCl or to water stress (24). Others using cells obtained from our laboratory, have confirmed our observation of the accumulation of this protein in salt adapted cells (10, 16). We have isolated osmotin from NaCl adapted tobacco cells in sufficient quantities for further characterization. In this report we describe a simple and rapid procedure for purification oftwo forms ofosmotin, some oftheir properties and localization within the cell, and the occurrence of significant amino acid sequence homology between osmotin, the sweet protein, thaumatin, and two other stress-related proteins.MATERIALS AND METHODS Cell Culture. Cell suspension cultures of tobacco (Nicotiana tabacum L. var Wisconsin-38) adapted to 25 g/L NaCl (S-25 cells) were maintained in our laboratory as described (12). These cells were harvested and stored as dry powders after washing in cold acetone (23

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A divalent metal ion-stimulated protein-serine/threonine phosphatase, PP1-arch, was purified approximately 1,000-fold from the extreme acidothermophilic archaeon Sulfolobus solfataricus (ATCC 35091). Purified preparations contained 40 to 70% of total protein as PP1-arch, as determined by assay-ing sodium dodecyl sulfatepolyacrylamide gels for protein phosphatase activity. The first 25 amino acids of the protein's sequence were identified, as well as an internal sequence spanning some 20 amino acids. Using this information, we cloned the gene for PP1-arch via the application of PCR and conventional cloning techniques. The gene for PP1-arch predicted a protein of 293 amino acids that bore striking resemblance to the members of the major family of protein-serine/threonine phosphatases from members of the domain Eucarya, the PP1/2A/2B superfamily. The core of the protein, spanning residues 4 to 275, possessed 29 to 31% identity with these eucaryal protein phosphatases. Of the 42 residues found to be absolutely conserved among the known eucaryal members of the PP1/2A/2B superfamily, 33 were present in PP1-arch. If highly conservative substitutions are included, this total reached 37. The great degree of sequence conservation between molecules from two distinct phylogenetic domains implies that the members of this enzyme superfamily had evolved as specialized, dedicated protein phosphatases prior to the divergence of members of the Archaea and Eucarya from one another.Protein phosphorylation-dephosphorylation represents a ubiquitous and prominent mechanism for regulating cellular processes. Protein kinases, protein phosphatases, and the phosphoproteins that they modify have been found in virtually every living organism. A priori, such universality might suggest that protein phosphorylation-dephosphorylation constitutes a very ancient means for exerting regulatory control. However, for many years, comparisons between the components of bacterial and eucaryal protein phosphorylation networks showed them to be structurally distinct, suggesting that each arose independently, at different times and places (reviewed in reference 18). For example, while all eucaryal protein kinases could be grouped into a single giant superfamily (14), the protein kinases from bacterial organisms showed no significant resemblance to this group (9). While members of the domain Eucarya phosphorylated some proteins on tyrosine residues, members of the domain Bacteria apparently did not (13). Therefore, it was and still is widely believed that protein phosphorylationdephosphorylation constitutes a relatively recent addition to the cell's regulatory repertoire, one that arose separately in each phylogenetic domain after it had diverged from its predecessors.Recent evidence has eroded the foundation upon which this consensus is based. Tyrosine-phosphorylated proteins have been detected in several members of the Bacteria (1,10,33,34,43). Genes encoding bacterial protein-histidine kinases have been discovered in eucaryal species ranging from rats (31...

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