Neutral Peptidases in the Stroma of Pea Chloroplasts

Abstract

ABSTRACIOne endopeptidase (EPI) and at least three aminopeptidases (API, AP2, and AP3) were discovered in the stroma of chloroplasts isolated from pea seedlings (Pisum sativum L.), and purified over 100-fold. EPI requires added Mg2" or Ca2" for activity, may have an additional tightly bound metal atom, and is inhibited by sulfhydryl reagents but not by serine residue-directed inhibitors. It is reversibly inhibited by dithiothreitol. Its specificity is for the bond between two adjacent Ala or Gly residues. Its molecular mass is 93 kilodaltons, estimated on a gel filtration column. Aminopeptidase activities were detected with the aid of different amino acyl-f-naphthylamides as substrates. They were resolved into at least three individual proteins by gel filtration and DEAE-cellulose chromatography, having apparent molecular masses of 269,000 (API), 84,000 (AP2), and 42,000 (AP3) daltons, respectively. Each has a unique specificity for substrates, with API hydrolyzing only the Prolyl-B-naphthylamide. None of the APs require added divalent cations for activity, but the possibility of a tightly bound metal function was suggested in AP2 and AP3 (not API) from effects of inhibitors. A probable sulfhydryl residue function was indicated for all three, from inhibition by p-hydroxymercuribenzoate and Zn2". All these peptidases had pH optima at 7.7.Specific protein turnover or selective protein degradation has been found to be an essential part of chloroplast development. In many cases there are reasons to think that the responsible proteases are located inside the chloroplasts. Proteolytic activity was postulated to be responsible for prolamellar body degradation when etiolated leaves are put in the light (5); for the removal of damaged QB apoprotein, and therefore for its high turnover rate (10, 15); for accelerated turnover of chloroplast-encoded 48 and 34.5 kD polypeptides in thylakoids lacking PSII (1 1); and for rapid degradation of NADPH:Pchlide reductase in barley etioplasts once the plants are put into the light (9). During leaf senescence, extensive protein degradations occurring in apparently intact chloroplasts are explained by an internal set of proteases (21). Chloroplast localization of peptidases was noted in wheat by Waters et al. (22). The best defined internal protease is the one in pea chloroplasts which processes ribulose 1,5-bis P carboxylase small subunit precursors imported from the cytosol; this was purified to a considerable degree ( 19).A thylakoid-located, ATP-dependent proteolytic activity was found responsible for removal of a significant fraction of newly translated proteins in pea chloroplasts, perhaps those which are mature but unassembled into larger complexes (12,14). On the other hand, an ATP-independent but Mg2+-dependent proteolytic activity seemed to be responsible for removal of newly translated proteins containing abnormal amino acids, and of prematurely terminated proteins (13). In an effort to locate the specific enzyme responsible for one or another of these activities, we have fo...

Modifications of Etioplasts in Cotyledons during Prolonged Dark Growth of Sugar Beet Seedlings (Identification of Etiolation-Related Plastidial Aminopeptidase Activities)

“…This degenerative process did not seem to be related to a decrease of production of plastidial proteins (see 'Results"). Previous reports had shown that ATP-independent endopeptidase activities were enhanced during natural senescence of leaves (Feller, 1986) and that ATP-independent neutral endopeptidases played an important role in plastids (Liu and Jagendorf, 1986;Musgrove et al, 1989;Bushnell et al, 1993), which may be compounded in a situation of decreased adenine nucleotide pool (El Amrani et al, 1994). In the present case, although a significant level of neutral ATP-independent endopeptidase activity was detected in purified plastids, the increase that was observed occurred after the onset of Glc limitation and loss of greening capacity.…”

Programmed cell death during plant growth and development

“…That plastids contains 460% of intracellular leaf protein suggests involvement of plastidic proteinases in remobilization of N during senescence (reviewed by Dalling and Nettleton, 1986). Chloroplastic ATP-dependent (Malek et al, 1984;Liu and Jagendorf, 1986) and non-ATP-dependent proteases (Bushnell et al, 1993) have been detected. However, one candidate for the ATP-dependent activity (the E. coli homolog ClpP system) is expressed constitutively and therefore does not appear to be a senescence-related protease (Shanklin et al, 1995).…”

Tansley Review No. 118

“…Proteases in the vacuole are capable of degrading Rubisco in vitro (Miyadai et al, 1990 ;Yoshida & Minimaka, 1996 ;Bose et al, 1999). However, it has been suggested that observations of chloroplast-associated protease activity (Liu & Jagendorf, 1986, Mae et al, 1989Bushnell et al, 1993) represent carry-over contamination (Bose et al, 1999). The persistence of the hypothesis that proteins must be translocated to lytic vacuoles for breakdown is largely due to the lack of chloroplast-targeting leader sequences on senescence-induced proteases, the apparent absence of ubiquitin, and the equivocal nature of the evidence from the ClpP protease system in the chloroplast.…”