InVitro Synthesis, Transport, and Assembly of the Constituent Polypeptides of the Light-Harvesting Chlorophyll a/b Protein Complex
Recent studies have established that transport across chloroplast envelopes of proteins which are synthesized by cytoplasmic ribosomes can occur by a post-translational mechanism (1,2). Dobberstein et al. (3) first discovered that a major chloroplast stromal protein, the small subunit (S) of ribulose 1,5-bisphosphate carboxylase (RuBPCase) is synthesized by free polysomes in the green alga, Chlamydomonas reinhardtii. Moreover, they found that translation of the small subunit messenger RNA in vitro yields a precursor (pS) 4000–5000 daltons larger than the mature protein. Upon incubation with a cell-free Chlamydomonas extract pS can be processed to the mature form and a small peptide fragment designated the transit peptide (4,5). Dobberstein et al. (3) proposed that transport of the RuBPCase small subunit in vivo occurs after it is completely synthesized and that the transit sequence on pS facilitates its post-translational interaction with the chloroplast envelope. This proposed mechanism is fundamentally distinct from the co-translational transport across endoplasmic reticulum membranes of proteins which are synthesized by membrane-bound ribosomes (6,7). Precursor forms of the RuBPCase small subunit also have been found among the translation products of spinach (1), pea (1,2,8) and duckweed (9) mRNA in cell-free systems.
KeywordsChlorophyll Urea Lithium Chloroform Electrophoresis
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