Synthesis of Thylakoid Membrane Proteins by Chloroplasts Isolated from Spinach

Intact chloroplasts, purified from spinach leaves by sedimentation in density gradients of colloidal silica, incorporate labeled amino acids into at least 16 different polypeptides of the thylakoid membranes, using light as the only source of energy . The thylakoid products of chloroplast translation were visualized by subjecting membranes purified from chloroplasts labeled with [88S]methionine to electrophoresis in high-resolution, SDS-containing acrylamide gradient slab gels and autoradiography . The apparent mol wt of the labeled products ranged from <10,000 to >70,000 . One of the labeled products is the apoprotein of the P700chlorophyll a-protein (CPI) . The CPI apoprotein is assembled into a pigmentprotein complex which is electrophoretically indistinguishable from the native CPI complex. Isolated spinach chloroplasts also incorporate [ 3H]leucine and [ 35S]methionine into cytochrome b559 . The radioactive label remains with the cytochrome through all stages of purification : extraction of the thylakoid membranes with Triton X-100 and urea, adsorption of impurities on DEAE cellulose, two cycles of electrophoresis in Triton-containing polyacrylamide gels and electrophoresis in SDS-containing gradient gels . Cytochrome b559 becomes labeled with both [3H]leucine and [35S]methionine and accounts for somewhat <1% of the total isotopic incorporation into thylakoid protein . The lipoprotein appears to be fully assembled during the time-course of our labeling experiments . Chloroplasts contain transcriptional and translational machinery which closely resembles that of bacterial cells and is distinct from that of the nuclear-cytoplasmic systems of higher plants and algae . Considerable effort has been directed for the past 15 years toward ascertaining the subcellular origins of chloroplast macromolecules and the contributions which the chloroplast makes to its own development and maintenance within the cell . One of the most fruitful approaches to the identification of chloroplast translation products J . CELL BIOLOGY © The Rockefeller University Press 0021-9525/80/05/0435/11 $1 .00 Volume 85 May 1980 435-445 has been to feed radioactive amino acids to isolated, intact chloroplasts followed by separation of the newly synthesized products by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE)' (reviewed in references 14 and 15) . By ' Abbreviations used in this paper: ALA, 5-aminolevulinic acid ; CF l, chloroplast coupling factor 1 ; CPI, the P700chlorophyll a-protein ; DTT, dithiothreitol; LA, levulinic acid ; PMSF, phenylmethyl sulfonyl fluoride ; SDSPAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis . 435 on A uust 8, 2017 jcb.rress.org D ow nladed fom use of autoradiography of two-dimensional slab gels, Ellis et al. (16) counted no fewer than 90 newly synthesized polypeptides in the stroma alone . Several of the polypeptides synthesized by isolated chloroplasts have been identified as specific proteins or as subunits of specific proteins . In the stromal fraction these include the large subunit of ribulose-1,5-bisphosphate carboxylase (3, 6, 33, 43) and the elongation factors EF-G and EF-T (42) ; in the thylakoid fraction, these include the a, a, and e subunits of chloroplast coupling factor 1 (CFI) (23) and cytochrome f(12) . A 30,00032,000 mol wt polypeptide of the thylakoids, also known as "peak D" (see, e .g ., reference 13), is widely recognized as the most rapidly labeled polypeptide in the thylakoid fraction, but it has not been identified beyond its mobility on SDS gels . In fact, most of the thylakoid polypeptides that are typically visualized by SDS-PAGE remain unidentified . In this study, we employed specific extractions and high-resolution gel electrophoresis techniques to characterize better and to determine the identities of some of the thylakoid polypeptides translated on ribosomes in isolated spinach chloroplasts . Our results support the general conclusions of other investigators who have either studied the synthetic activities of isolated plastids (6, 13, 20, 21, 33, 43) or examined the synthesis of plastid proteins under rigorously controlled conditions of site-specific inhibition of protein synthesis in vivo (9) . More importantly, our results extend these earlier studies by identifying two specific, integral thylakoid polypeptides, the apoproteins of cytochrome b559 and the P700-chlorophyll a-protein (CPI), as translation products of the plastid . In addition, we present evidence that suggests that these two apoproteins become associated with their respective porphyrin prosthetic moieties during the time-course of our experiments . Preliminary accounts of parts of this work have been presented elsewhere (47, 48) . MATERIALS AND METHODS