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

Abstract

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

Characterization of Thylakoid-Derived Lipid-Protein Particles Bearing the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

“…However, it seems clear that at least a portion of the RLSU is synthesized on thylakoid-bound polysomes and may associate with thylakoids before incorporation into Rubisco holoenzyme (Bhaya and Jagendorf, 1984;Hattori and Margulies, 1986;Makino and Osmond, 1991). There is also evidence that thylakoid-bound polysomes engage in cotranslational insertion into thylakoids Kim et al, 1991).…”

“…Large subunit mRNA (rbcL) also has sequence motifs in common with the mRNAs of some chloroplastically encoded thylakoid proteins, including the /3-subunit of ATP synthase (atpB), the core complex of PSI (psaA), and the D1 protein of PSII (@A) (Kim et al, 1993). In addition, RLSU, like chloroplastically encoded thylakoid proteins, appears to be translated by thylakoid-bound polysomes associated with stromal lamellae and the outer margins of grana1 stacks (Yamamoto et al, 1981;Bhaya and Jagendorf, 1984;Hattori and Margulies, 1986;Klein et al, 1988).…”

“…It has been demonstrated that at least a portion of RLSU synthesis occurs on thylakoid-bound polysomes of spinach and pea chloroplasts (Bhaya and Jagendorf, 1984;Hattori and Margulies, 1986). These polysomes have been implicated in the co-translational insertion of thylakoid-localized proteins encoded by ctDNA (Chua et al, 1973;Margulies et al, 1975;Michaels and Margulies, 1975;Minami and Watanabe, 1984;Kim et al, 1991).…”

Further Characterization of Ribosome Binding to Thylakoid Membranes

“…The ribosomes are bound in part by saltdissociable electrostatic forces, and in part by nascent chains, since the combination of puromycin and high salt releases twice as many as high salt alone does (16,24). In a study where high incorporation rates were obtained using pea thylakoids as many as 30 labeled protein bands were detected on SDS gel electrophoresis (3). Specific translation products identified as made by thylakoid-bound ribosomes include a polypeptide of PSII (11), the 32 kD QB apoprotein (17), Cytf (23), the a and d subunits of CF1 (5,17), subunit III of CFo (4), and the PSI reaction center polypeptides (15).…”

“…Protein synthesis by isolated chloroplasts, was performed at a light intensity of 45 umol/m2.s' as described (19); except that the concentration of (3H]Leu in the medium was 2 uCi/ml at a specific activity of 151 gCi/nmol instead of 0.1 1 to 46 gCi/nmol. Protein synthesis by thylakoids was performed as described previously (3).…”