Distribution of Several Proteases Inside and Outside the Central Vacuole of Chara australis

Transcription

1 CELL STRUCTURE AND FUNCTION 11, (1986) by Japan Society for Cell Biology Distribution of Several Proteases Inside and Outside the Central Vacuole of Chara australis Yuji Moriyasu and Masashi Tazawa Department of Biology, Faculty of Science, University of Tokyo, Hongo, Tokyo 113, Japan ABSTRACT. Vacuolar sap was separated by intracellular perfusion and the distribution of proteases was studied in the giant alga Chara australis. Caseinolytic and hemoglobin-digesting activities were found to be higher at low ph, and about 85 % of total activity was localized in the central vacuole. The optimal ph for carboxypeptidase activity measured using N-carbobenzoxy-L-phenylalanyl-L-alanine as a substrate was around 5, and about 95 % of total activity was localized in the vacuole. Moreover a substantial portion (40 %) of aminopeptidase activity, measured at ph 5.5 using L- leucine-Ĉ-naphthylamide as a substrate, was found in the vacuole. It is known that most cellular proteins turn over (5, 6, 8, 9). In animal cells, lysosomes are assumed to play a major role in the degradative processes which contribute to protein turnover. Matile (14) proposed that plant vacuoles correspond to animal lysosomes and are lytic compartments for macromolecules. This hypothesis is based on two types of observation. One is that the vacuole contains various hydrolytic enzymes, and the other is that cytoplasmic invaginations into the vacuole have been observed in electron microscopic studies (4, 18). In previous paper (16), we investigated the mechanism of vacuolar ph regulation in internodal cells of Characeae, and found that the vacuolar ph is maintained almost constant against changes in environmental conditions and experimental perturbations. According to Matile's theory (14), one of physiological roles of vacuolar ph regulation may be the maintenance of suitable conditions for hydrolytic enzymes with ph optima in the acidic region. Thus, we investigated the vacuolar/extravacuolar distribution of several protease activities, using internodal cells of Chara australis, in which we could easily separate the vacuolar sap from the extravacuolar compartment. The presence of carboxypeptidase in the vacuole of Chara internodal cells has already been reported by Doi et al. (3). MATERIALS AND METHODS Plant Materials. Chara australis was cultured as described earlier (16). For the fractionation of vacuolar sap, internodal cells, 5-15 cm in length and 500-1,000 Đm in diameter, were used. Chemicals. All chemicals used were of analytical grade. Casein (according to Hammarsten) was purchased from E. Merck, Darmstadt, Germany. Bovine blood hemoglobin, type I and N-carbobenzoxy-L-phenylalanyl-L-alanine (CBZ-Phe-Ala) were obtained 81

2 82 Y. Moriyasu and M. Tazawa from Sigma Chemical Company, U.S.A. L-leucine-ƒÀ-naphthylamide hydrochloride (Leu-(ƒÀnaphthylamide), L-leucine-p-nitroanilide hydrochloride (Leu-p-nitroanilide) and N- tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS) were obtained from Wako Pure Chemical Industries, Ltd., Japan. Preparation of enzyme solution. Chara australis was homogenized in a mortar and pestle with 10 mm phosphate-na, ph 7.0 containing 1 mm 2-mercaptoethanol. The homogenate was filtered through one sheet of gauze and centrifuged at 10,000 rpm (8,000 g) for 10 min. The supernatant was used as a crude enzyme solution except when ninhydrin reaction was used for enzyme assay. When ninhydrin was used, the supernatant solution was passed through a Sephadex G-25 column which was preequilibrated with 10 mm phosphate-na, ph 7.0 for desalting. Cell fractionation. For investigating the vacuolar/extravacuolar distribution of protease activities, cell fractionation was performed as follows. Internodal cells were placed on a polyacrylate bench. When the turgor disappeared due to transpiration from the cell surface, both cell ends were amputated using scissors and the vacuole was perfused with a solution consisting of 80 mm KC1, 30 mm NaCl, 10 mm CaCl2 and 10 mm MgCl2. The perfusate was used as the vacuolar sap fraction. The remaining portion was used for preparing the enzyme solution from the extravacuolar fraction. Enzyme assays. Caseinolytic and hemoglobin-digesting activities were measured as increases in trichloroacetic acid soluble, ninhydrin-positive substances. The reaction mixture contained enzyme solution, 100 mm buffer of various phs, 1 mm 2-mercaptoethanol and 0.5 % (w/v) casein or 0.25 % (w/v) hemoglobin. The reaction was started by adding substrate, and the mixture was incubated at 37 Ž with shaking. At regular time intervals, an aliquot of the mixed solution was taken out and the reaction stopped by adding trichloroacetic acid (final concentration 5 %). After centrifuging the mixture at 16,000 rpm for 10 min, the supernatant was tested for ninhydrin reaction (15). Carboxypeptidase activity was assayed using CBZ-Phe-Ala as a substrate (3). The reaction mixture contained enzyme solution, 100 mm buffer of various phs, 1 mm 2-mercaptoethanol and 1 mm CBZ-Phe-Ala. The reaction was carried out at 37 Ž with shaking. At regular time intervals, an aliquot was taken out and the time dependent increase in released Ala was measured by ninhydrin reaction. Aminopeptidase activities were assayed using two kinds of substrates. In the case of Leu-ƒÀ-naphthylamide, the reaction mixture contained enzyme solution, 100 mm buffer of various phs and 1 mm Leu-/9-naphthylamide. The reaction was started by adding the substrate, carried out at 37 Ž with shaking and stopped by adding 0.1 % (w/v) Fast Garnet GBC salt in 1 M acetate-na, ph 4.2, containing 10 % (v/v) Tween 20 (10). Released ƒànaphthylamine was measured by color development at A525 using a spectrophotometer (Hitachi 220A Spectrophotometer, Hitachi, Ltd., Tokyo, Japan). In the case of Leu-pnitroanilide, the reaction mixture contained enzyme solution, 100 mm buffer of various phs and 1 mm Leu-p-nitroanilide. P-Nitroaniline was monitored as an increase in A410 using a spectrophotometer. One unit of the enzymes corresponds to the hydrolysis of 1 ƒêmol of substrates per minute. In the case of caseinolytic and hemoglobin-digesting activities, 1 unit is defined as the production of hydrolysate equivalent to 1 ƒêmol of alanine by ninhydrin reaction per minute. RESULTS Fig. 1 a, b shows the dependencies of caseinolytic (a) and hemoglobin-digesting (b)

3 Vacuole/extravacuole Distribution of Proteases 83 Fig. 1. Effect of ph on caseinolytic (a), hemoglobin-digesting (b), carboxypeptidase (c), L- leucine-Ĉ-naphthylamidase (d) and L-leucine-p-nitroanilidase (e) activities in crude extract from Chara cells. Carboxypeptidase activity was measured using N-carbobenzoxy-L-phenylalanyl-L-alanine as a substrate. 100 mm citrate-na (ph 3.0 to 6.5), 100 mm phosphate-na (ph 6.5 to 8.0) or 100 mm TAPS-Na (ph 8.0 to 9.0) were used as buffers for enzyme assay.

4 84 Y. Moriyasu and M. Tazawa TABLE 1. VACUOLAR/EXTRAVACUOLAR DISTRIBUTION OF PROTEASES IN CHARA INTERNODAL CELLṢ activities on ph. A higher caseinolytic activity was detected in the acidic region (near ph 5). Hemoglobin-digesting activity was also conspicuous in the acidic region and the optimal ph was lower than for caseinolytic activity. Carboxypeptidase activity is plotted against ph in Fig. lc. The optimal ph was near 5, the natural ph of vacuolar sap in Chara australis (16). This result agrees with that of Doi et al. (3). Fig. 1 d shows the dependence of leucine-ƒà-naphthylamidase activity on ph. Its optimal activity existed between 5.5 to 6.0. When the ph was less than 4.0, no activity was measured, whereas in the alkaline region a significant activity was observed. Fig. le shows the ph-dependence of the leucine-p-nitroanilidase activity. When the ph was less than 4.5, no activity was detected, but in the alkaline region a broad optimum was observed. To investigate the localization of these proteolytic activities, internodal cells were separated into vacuolar sap and a remaining portion. Table 1 shows that about 85 % of caseinolytic and hemoglobin-digesting activities having ph optima in the acidic region were found in the central vacuole. Carboxypeptidase activity was also mostly vacuolar. In contrast, it was difficult to localize aminopeptidase activities. In the alkaline region (ph 8.0), % of both leucine-ƒà-naphthylamidase and leucine-pnitroanilidase were extravacuolar. In the acidic region (ph 5.0), more than half of the leucine-ƒà-naphthylamidase activity was detected in the extravacuolar compartment. DISCUSSION We found that several proteases which hydrolyze artificial substrates such as CBZ- Phe-Ala and exogenous proteins such as hemoglobin or casein are localized in the central vacuole of Chara australis. Vacuolar localization of hydrolytic enzymes, especially proteases, has been reported by many workers (1, 2, 7, 11, 17, 19, 20, 21). In higher plants, the vacuolar/extravacuolar distribution of proteases is determined using isolated vacuoles which are often prepared from protoplasts. There are two problems with this method. Firstly, it is difficult to separate the activities of foreign proteases used for the isolation of protoplasts from the activities of native proteases. Secondly, the yield of the vacuoles from protoplasts is less than 100 %. Therefore, calculating the vacuolar/extravacuolar distribution of protease activities is based on

5 Vacuole/extravacuole Distribution of Proteases 85 the assumption that one plant cell contains one vacuole. This inevitably causes some error. In giant algal cells, such as Acetabularia or Chara, from which the vacuolar sap or the vacuole-rich fraction is easily prepared by mechanical operation (3, 12), the vacuolar localization of hydrolytic enzymes has been reported. In Chara cells, the localization of carboxypeptidase in the central vacuole was reported by Doi et al. (3). This was reconfirmed by the present study. In many plants, cytoplasmic invaginations into the vacuole and cytoplasmic drops in the vacuole have been observed in electron micrography (4, 18). Vacuolar proteases have broad specificities and can nonspecifically degrade proteins in cytoplasmic drops to produce amino acids, which can then be used for protein synthesis. These processes seem to occur mainly under stressed conditions or during senescence. In the normal state, cytoplasmic proteins have individual turnover rates. If vacuolar proteases are involved in protein turnover in the normal state, the selection of proteins to be degraded should occur not in the vacuole but in the cytoplasm. In animal cells, a non-lysosomal pathway for protein breakdown has been discussed (5, 6, 8). In higher plants, it has been proposed that the initial degradation of ribulose bisphosphate carboxylase under stressed conditions occurs inside chloroplasts (13). In Chara australis, about 75 % of assayable aminopeptidase activity was extravacuolar. Further localization of aminopeptidases in the cytoplasm is under investigation. To elucidate the mechanism of intracellular protein breakdown, it is necessary to investigate the detailed localization of all protein degrading enzymes. Acknowledgments. This work was partly supported by Special Coordination Funds for the Promotion of Science and Technology from the Science and Technology Agency of Japan. We wish to thank Dr. K. Sakano and Dr. A. Okitani for helpful suggestions. REFERENCES 1. BOLLER, T. and H. KENDE. Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol. 63, , CANUT, H., G. ALIBERT and A.M. BOUDET. Proteases of Melilotus alba mesophyll protoplasts. I. Intracellular localization. Plant Science 39, , Doi, E., C. OHTSURU and T. MATOBA. Lysosomal nature of plant vacuoles. II. Acid hydrolases in the central vacuole of internodal cells of Charophyta. Plant Cell Physiol. 16, , FINERAN, B.A. Organization of the tonoplast frozen-etched root tips. J. Ultrastruct. Res. 35, , GOLDBERG, A.L. and J.F. DICE. Intracellular protein degradation in mammalian and bacterial cells. Ann. Rev. Biochem. 43, , GOLDBERG, A.L. and A.C. ST. JOHN. Intracellular protein degradation in mammalian and bacterial cells : Part 2. Ann. Rev. Biochem. 45, , HECK, U., E. MARTINOIA and P. MATILE. Subcellular localization of acid proteinase in barley mesophyll protoplasts. Planta 151, , HERSHKO, A. and A. CIECHANOVER. Mechanisms of intracellular protein breakdown. Ann. Rev. Biochem. 51, , HUFFAKER, R.C. and L.W. PETERSON. Protein turnover in plants and possible means of its regulation. Ann. Rev. Plant Physiol. 25, , KOLEHMAINEN, L. and J. MIKOLA. Partial purification and enzymatic properties of an aminopeptidase from barley. Arch. Biochem. Biophys. 145, , LIN, W. and V.A. WITTENBACH. Subcellular localization of proteases in wheat and corn mesophyll protoplasts. Plant Physiol. 67, , LUSCHER, A. and P. MATILE. Distribution of acid ribonuclease and other enzymes in stratified

6 86 Y. Moriyasu and M. Tazawa Acetabularia. Planta 118, , MAE, T., N. KAI, A. MAKINO and K. OHIRA. Relation between ribulose bisphosphate carboxylase content and chloroplast number in naturally senescing primary leaves of wheat. Plant Cell Physiol. 25, , MATILE, P. The lytic compartment of plant cells. Cell Biology Monographs, Vol. 1, Springer- Verlag, Wien New York, MOORE, S. and W.H. STEIN. A modified ninhydrin reagent for the photometric determination of amino acids and related compounds. J. Biol. Chem. 211, , MORIYASU, Y., T. SHIMMEN and M. TAZAWA. Vacuolar ph regulation in Chara australis. Cell Struct. and Funct. 9, , NISHIMURA, M. and H. BEEVERS. Hydrolases in vacuoles from castor bean endosperm. Plant Physiol. 62, 44-48, Poux, N. Sur la presence d'enclaves cytoplasmiques in voie de degenerescence dans les vacuoles des cellules vegetales. C.R. Acad. Sci. (Paris) 257, , WAGNER, G.J., P. MULREADY and J. CUTT. Vacuole/extravacuole distribution of soluble protease in Hippeastrum petal and Triticum leaf protoplasts. Plant Physiol. 68, , WATERS, S.P., E.R. NOBLE and M.J. DALLING. Intracellular localization of peptide hydrolases in wheat (Triticum aestivum) leaves. Plant Physiol. 69, , WITTENBACH, V.A., W. LIN and R.R. HEBERT. Vacuolar localization of proteases and degradation of chloroplasts in mesophyll protoplasts from senescing primary wheat leaves. Plant Physiol. 69, , 1982 (Received for publication, January 10, 1986)