Factors Permitting Prolonged Translation by Isolated Pea Chloroplasts

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ABSTRACIPolysomes bound to washed thylakoids from pea Pisum sativum cv Progress No. 9 chloroplasts are capable of protein synthesis when supplemented with amino acids, ATP and a regenerating system, GTP, and soluble factors required for translation. The extent of protein synthesis in previous reports, however, was quite low when compared to in organeilo transltion. By systematic testing of parameters in the isolation of thylakoids and reaction mixture components we have been able to establish more optimal conditions. Incorporation of 2 to 10 nanomoles of leucine per milligram chlorophyll in a 20-minute reaction period is now possible, representing a 10-to 60-fold increase over amounts previously reported. Autoradiographs of solubilized, electrophoresed membranes show about 30 discrete labeled polypeptides which remain associated with the thylakoid membranes.There are two populations of ribosomes within chloroplastthose free in the stroma, and those bound to the thylakoid membranes (20,27). Further, it is known that a large fraction of the bound ribosomes are in the form of polysomes attached at least in part by nascent peptide chains (3,20,21,27). These attached polysomes can continue protein synthesis when supplied with ATP, GTP, amino acids, and soluble factors (enzymes and tRNAs) for translation, although at quite low rates (3, 21) compared to those found in organello (12,22). Before investigating the nature of polypeptides synthesized by attached polysomes, we felt it important to optimize the conditions for translation, since a distorted picture of the extent and relative abundance of specific polypeptides may result from nonoptimal reaction rates (17

Section: Resultsmentioning

confidence: 91%

mentioning

confidence: 99%

Optimal Conditions for Translation by Thylakoid-Bound Polysomes from Pea Chloroplasts

Bhaya

Jagendorf²

1984

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“…The chloroplast protein synthesizing system used here has been optimized with respect to the rate and duration ofamino acid incorporation into protein (8,17). The extracts appeared to be unable to support incorporation of all newly synthesized large subunits into RuBisCO, even when excess small subunits were added.…”

Section: Materuils and Methodsmentioning

confidence: 99%

Incorporation of Large Subunits into Ribulose Bisphosphate Carboxylase in Chloroplast Extracts

Roy¹,

Chaudhari²,

Cannon³

1988

ABSTRACTbulk of the large subunits, however, occur in association with a 29S binding protein complex (3,5,18). ATP brings about the dissociation of this complex into 7S complexes and monomeric (about 60 kD) binding protein subunits (5,11,15). Recently our laboratory has shown that antibody directed against this binding protein inhibits the in vitro incorporation of 7S large subunits into RuBisCO (6). It was also found that very low concentrations of ATP appear to support a reaction which limits the incorporation of large subunits into RuBisCO (6).The incorporation of large subunits into RuBisCO could be due to de novo assembly or it could reflect some kind ofexchange reaction. If the incorporation is due to de novo assembly, then it should be dependent on small subunits; data presented here indicate that added small subunits can stimulate the incorporation of large subunits into RuBisCO in chloroplast extracts.We observed an upper limit to the ability of added small subunits to stimulate RuBisCO assembly. Searching for limiting factors, we investigated the effect of pH and sorbitol concentration during in organello protein synthesis on subsequent in vitro assembly of large subunits into RuBisCO. Significant effects of sorbitol concentration were observed, indicating that optimal in vitro assembly was obtained after large subunits were synthesized in chloroplasts under hypotonic conditions. MATERUILS AND METHODSRuBisCO2 (EC 4.1.1.39) catalyzes the CO2 fixation step in the Calvin cycle of photosynthesis. It also catalyzes the competing 02 fixation step in photorespiration (12). The enzyme consists of large subunits encoded in chloroplast DNA (13) and small subunits encoded in nuclear DNA. The small subunits are synthesized as precursors, taken up and processed by chloroplasts, and assembled with large subunits into RuBisCO in the chloroplast stroma (7). The mechanism of these final assembly steps appears to involve a large subunit binding protein, which is not part of the final structure of the enzyme (3,5,6,9,(14)(15)(16)18).The large subunits which are incorporated into RuBisCO in pea chloroplast extracts are derived from 7S complexes (15).

“…9, Agway, Rochester, NY), were grown on a 12 h light/dark cycle as described previously (4 Figure 1: A, 0°C-translation at 188 mm sorbitol; B, optimal assembly conditions, translation at 188 mM sorbitol; C, 0°C-translation at 377 mm sorbitol; D, optimal assembly conditions, translation at 377 mm sorbitol. (18), and illuminated in the presence of [35S]methionine (>1000 Ci/mmol, New England Nuclear Corporation, Waltham, MA).…”

Section: Growth Of Plantsmentioning

confidence: 99%

Delayed Osmotic Effect on in Vitro Assembly of RuBisCO

Chaudhari¹,

Roy²

1989

Higher plant ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) cannot reassociate after dissociation, and its subunits do not assemble into active RuBisCO when synthesized in Escherichia coil. Newly synthesized subunits of RuBisCO are associated with a high molecular weight binding protein complex in pea chloroplasts. The immediate donor for large subunits which assemble into RuBisCO is a low molecular weight complex which may be derived from the high molecular weight binding protein complex. When the high molecular weight binding protein complex is diluted, it tends to dissociate, forming low molecular weight complexes. When the large subunit-binding protein complexes were examined after in organello protein synthesis, it was found that the low molecular weight complexes were more abundant when protein synthesis was carried out under hypotonic conditions. This increase in the assembly competent population of low molecular weight large subunit complexes can account for the increased amount of In vftro RuBisCO assembly which occurs under these conditions. The data indicate that the assembly of large subunits into RuBisCO is a function of the aggregation state of the large subunit binding protein complex during protein synthesis. This implies that the binding protein exerts its effects during or shortly after large subunit synthesis.