Intracellular Localization of Peptide Hydrolases in Wheat ( Triticum aestivum L.) Leaves

Endoproteinase activities and species were compared during darkinduced senescence of attached and detached primary barley leaves by isoelectric focusing and polyacrylamide gel electrophoresis of cell-free extracts. Neither of the two major endoproteinases (EPI and EP2) changed in amounts during senescence of attached leaves, nor did new endoproteinases appear. In contrast, during senescence of detached leaves, both EPI and EP2 activities increased and four new species of endoproteinases appeared. Proteolytic activity was evenly distributed throughout attached leaves, but activity in the detached leaf increased sharply from the tip to the base with the four new higher molecular weight species of proteinases present only in the bottom half of the leaf nearest the cut end. Thus a wound response may be superimposed on the processes of senescence in detached leaves. Cycloheximide and kinetin both inhibited the increase of EPI, EP2, and the induction of the four new endoproteinases; chloramphenicol had no effect. Indications are that both the increases in activity and the induction of new species of proteinases were the result of activity of cytoplasmic ribosomes.Hydrolysis of total protein and ribulose-1,5-bisphosphate carboxyase protein in vivo was somewhat faster in detached than attached leaves. The difference, however, was much less than would be expected from the great increase in proteolytic activity in detached leaves.Large-scale hydrolysis of proteins is an event occurring early in leaf senescence. Large increases in proteolytic activity occur in detached oat (17), barley (25), and corn (7)

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Differential Induction of Endoproteinases during Senescence of Attached and Detached Barley Leaves

Miller¹,

Huffaker²

1985

“…Isolated chloroplasts from barley (3) and soybean (19) were capable of degrading RuBPCase4; an ATP-dependent proteolytic activity was located in thylakoids from pea chloroplasts (8,11), and one endoprotease and three APs were found in the stromal fraction of pea chloroplasts (9); endoproteolytic activity against RuBPCase was found in the stromal fractions of chloroplasts from barley leaves (22,23) Nettleton et al (17) and Tang and Huffaker (23) found proteolytic activities associated with the thylakoid fractions of chloroplasts from wheat and barley leaves, respectively, capable of degrading RuBPCase. In wheat, 50% of the AP activity was localized in chloroplasts with the remainder in the cytoplasm (30).…”

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confidence: 99%

“…AP activity was assayed spectrophotometrically (20,30) with each assay consisting of 0.8 ml of 12.5 mm K-phosphate buffer (pH 6.8), 0.1 ml of 10 mM L-aa-/3-NA substrate, and 0.1 ml of enzyme source (total volume, 1 ml). NADPH-dependent TPDH was assayed according to Heber et aL (6).…”

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confidence: 99%

Characterization and Subcellular Localization of Aminopeptidases in Senescing Barley Leaves

Thayer¹,

Choe²,

Rausser³

et al. 1988

ABSTRACIFour aminopeptidases (APs) were separated using native polyacrylamide gel electrophoresis of cell-free extracts and the stromal fractions of isolated chloroplasts prepared from primary barley (Hordeum vulgare L., var Numar) leaves. Activities were identified using a series of aminoacyl-,B-naphthylamide derivatives as substrates. AP1, 2, and 3 were found in the stromal fraction of isolated chloroplasts with respective molecular masses of 66.7, 56.5, and 54.6 kilodaltons. AP4 was found only in the cytoplasmic fraction. No AP activity was found in vacuoles of these leaves. It was found that 50% of the L-Leu-#-naphthylamide and 25% of the L-Arg-ji-naphthylamide activities were localized in the chloroplasts. Several AP activities were associated with the membranes of the thylakoid fraction of isolated chloroplasts. API, 2, and 4 reacted against a broad range of substrates, whereas AP3 hydrolyzed only LArg-B-naphthylamide. Only AP2 hydrolyzed L-Val-a-naphthylamide. Since AP2 and AP3 were the only ones reacting against Val-#-naphthylamide and Arg-8-naphthylamide, respectively, several protease inhibitors were tested against these substrates using a stromal fraction from isolated chloroplasts as the source of the two APs. Both APs were sensitive to both metallo and sulfhydryl type inhibitors. Although AP activity decreased as leaves senesced, no new APs appeared on gels during senescence and none disappeared.Soluble proteins are rapidly degraded or mobilized in the early phases of leaf senescence. Chloroplast proteins are among the first to be degraded, and chloroplast constituents disappear faster than the chloroplast organelles (10,13,28). This suggests that chloroplasts contain proteolytic activities capable of degrading their protein constituents. Such proteolytic activities in chloroplasts have, in fact, been found. Isolated chloroplasts from barley (3) and soybean (19) were capable of degrading RuBPCase4; an ATP-dependent proteolytic activity was located in thylakoids from pea chloroplasts (8, 11), and one endoprotease and three APs were found in the stromal fraction of pea chloroplasts (9); endoproteolytic activity against RuBPCase was found in the stromal fractions of chloroplasts from barley leaves (22,23) Nettleton et al. (17) and Tang and Huffaker (23) found proteolytic activities associated with the thylakoid fractions of chloroplasts from wheat and barley leaves, respectively, capable of degrading RuBPCase. In wheat, 50% of the AP activity was localized in chloroplasts with the remainder in the cytoplasm (30).To better understand the degradation ofchloroplastic proteins, it is necessary to identify and characterize the proteases and their subcellular localization. Recent information shows that the in vivo half-life of some bacterial proteins is a function of their amino terminal amino acid residue (2). If future work shows this applicable to plants, APs may be important in the degradation of chloroplast protein. In this paper, we have identified four APs from barley leaves that are localized in both...

“…The vacuoles are the main site of protease deposition (3,6,10,18,25,(30)(31)(32). This conspicuous cell compartment was shown to be directly involved in the breakdown of storage proteins during germination in seeds (24) (19,20) and to contain a specific protease directed towards the light subunit of RuBP carboxylase (27).…”

Section: Introductionmentioning

confidence: 99%

Hydrolysis of Intracellular Proteins in Vacuoles Isolated from Acer pseudoplatanus L. Cells

Canut¹,

Alibert²,

Böudet³

1985

Acerpewdoepaxas cell suspension cultures were used to examine the ability of vacuoles isolated from protoplasts to hydrolyze their endogenous proteins. Total cell proteins were labeled by addition of VIHlleucine to the culture medium. After preparation of the protoplasts, vacuoles were isolated and were shown to be essentially free from other cellular components. Up to 30% of the IHlleucine-labeled newly synthesized proteins were recovered in the vacuoles. When incubated for 6 hours at 20°C, the vacuoles degraded half of these proteins. The protein breakdown was temperature and pH dependent. Analysis by electrophoresis, in denaturing polyacrylamide gels, revealed that most of the vacuolar proteins were degraded. However, some vacuolar proteins were unaffected during a 6-hour incubation period. The results indicate that vacuoles are able to acquire and degrade intracellular proteins.In higher plant cells, intracellular protein breakdown is a fundamental process which has important implications in biochemical regulation and physiological events such as protein turnover, germination, or senescence (7,16,23,28). However, the mechanisms that control the degradation of proteins are still obscure and the most complete data available today concern the characterization of proteases and particularly their intracellular location.The vacuoles are the main site of protease deposition (3,6,10,18,25,(30)(31)(32). This conspicuous cell compartment was shown to be directly involved in the breakdown of storage proteins during germination in seeds (24) (19,20) and to contain a specific protease directed towards the light subunit of RuBP carboxylase (27). So, it is now thought that the vacuole is not the only site of protein degradation in the plant cell, and the question arising is: are vacuoles only involved in particular physiological events such as germination or senescence, or are they also involved in continuous protein turnover?In this paper, we present evidence that isolated vacuoles from growing cells from Acer pseudoplatanus suspension cultures are able to degrade their endogenous proteins. ' Supported by grants from the Centre National de la Recherche Scientifique and from the Universite Paul Sabatier Toulouse. MATERIALS AND METHODSCell Suspension Cultures of Sycamore. The cells were grown on the liquid medium of Lamport (15) al. (1). This procedure allows, in one-step, the lysis of the protoplast plasmalemma and the rapid separation (less than 1 min) of the released vacuoles from the other compartments of the protoplast. Vacuole yields and contamination of the preparations by other organelles were similar to those described previously (1).Labeling of Cell Proteins. Fifty MCi [3H]leucine (45 Ci/mmol, C.E.A., France) in 4 ml culture medium was injected aseptically through the culture vial cotton wool plug of7-d-old cells. Usually, the incorporation proceeded for 18 h, then the cells were harvested and protoplasts and vacuoles prepared as described above.Precipitation of Proteins. The protoplasts or the vacuoles (...