Suspension-cultured cells of tomato (Lycopersicon esculentum) start to secrete an RNA-degrading enzyme activity during transition from logarithmic to stationary growth phase. Using affinity chromatography on agarose-5-(4-aminophenyl-phosphoryl) uridine 3'(2') monophosphate as a powerful and final enrichment step, the enzyme was purified to homogeneity and characterized as ribonuclease I (RNase I) according to the following data: (a) it has an Mr of 22,000 (sodium dodecyl sulfatepolyacrylamide gel electrophoresis), a pH-optimum of pH 5.5, a pi of 3.9, and its activity was found to be insensitive to EDTA; (b) the enzyme splits single-stranded RNA endonucleolytically by a phosphotransferase reaction yielding 2',3'-cNMPs as primary monomeric products; (c) as studied with diribonucleoside monophosphates as substrates, the enzyme exhibits a pronounced preference for 5' purine residues adjacent to the cleavage site.
The primary structure of an extracellular ribonuclease (RNase LE) from Pi-depleted media of cultured cells of Lycopersicon esculenturn L. cv. Lukullus has been determined. This was carried out by analysis of peptides isolated after enzymatic and chemical cleavage of the reduced and S-ethylpyridylated protein.RNase LE consists of 205 amino acid residues and has a molecular mass of 22 666 Da and an isoelectric point of 4.24. The enzyme contains 10 half-cystines. There are no potential N-glycosylation sites in the sequence.The sequence of RNase LE is homologous with those of self-incompatibility proteins of several higher plant species and with those of a number of fungal RNases. The sequence similarity with the family of self-incompatility proteins is greater than with the fungal RNases, suggesting that the self-incompatibility proteins arose from ancestral RNase by gene duplication after the divergence of higher plants and fungi. Two pentapeptide sequences, i. e. HGLWP and KHGTC (or KHGSC), are present at identical positions in all the aligned proteins, suggesting that they contribute to the active site.RNA metabolism, which includes processing, turnover and degradation of cellular RNA, has emerged as a complex and important determinant of gene expression in living cells. Much of our current knowledge on enzymes hydrolyzing RNA (RNases) and their in vivo functions comes from studies with Escherichia coli [l]. With respect to structural characterization and regulation of RNases from plant origin, knowledge lags far behind that already well established for the enzymes from fungi [2] and mammals [3].A number of physiological functions in plants were found to be associated with changes in nucleolytic activities (see [4] and [5] for reviews). More recent studies on nucleolytic and ribonucleolytic enzymes have shown that their expression is regulated by hormones [6], light and senescence [7], plant development [8], aging and wounding [9] or nutrient starvation [lo, 111 (see below). Investigation of these enzymes may lead to the identification of regulatory elements and signals for plant gene expression in that field.
Four intracellular RNases were found to be induced in cultured tomato (Lycopersicon esculentum) cells upon phosphate starvation. Localization studies revealed three (RNases LV 1-3) in the vacuoles and one (RNase LX) outside these organelles. All of these RNases were purified to homogeneity and were shown to be type I RNases on the basis of type of splitting, substrate, and base specificity at the cleavage site, molecular weight, isoelectric point, and pH optimum. Moreover, RNase LV 3 was shown by fingerprinting of tryptic digests on reversed-phase high-performance liquid chromatography and sequencing the N terminus and two tryptic peptides to be structurally very similar to a recently characterized extracellular RNase LE which is also phosphate regulated (Nürnberger et al. [1990] Plant Physiol 92: 970-976; Jost et al. [1991] Eur J Biochem 198: 1-6). Expression of the four intracellular RNases is induced by depleting the cells of phosphate and repressed by adding phosphate. Our studies indicate that higher plants, in addition to secreting enzymes for scavanging phosphate under starvation conditions, also induce intracellularly emergency rescue systems.
In previous work we have determined the primary structure of two of the five ribonucleases which are induced by phosphate starvation in cultured tomato cells. Here, we present the isolation and characterization of the cDNAs for the extracellular ribonuclease LE and the intracellular, but extravacuolar ribonuclease LX. Structural analysis of these cDNAs together with partial protein-sequencing of vacuolar ribonucleases LV1, LV2 and LV3 revealed a family of very similar ribonucleases. From these data we assume identify between ribonucleases LE and LV3 for which the targeting mechanism has to be shown. Furthermore, RNase LV1 and RNase LV2 might be posttranslational processing products of RNase LX which travel to the vacuoles after splitting off the putative ER retention signal present at RNase LX. Additionally, we show by northern blot analysis that phosphate starvation in plant cells leads to an increase in the steady-state level of this type of enzymes revealing close similarities of the plant response to a limited supply of inorganic phosphate with the PHO regulation in bacteria and fungi.
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