Student Theses and Dissertations
Date of Award
1979
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
Nam-Hai Chua
Keywords
chloroplast protein synthesis, Chlamydomonas reinhardtii, thylakoid membranes, in vivo labeling, integral membrane proteins
Abstract
Protein synthesis in the Chlamydomonas reinhardtii chloroplast was investigated by in vivo and in vitro approaches. The site of synthesis of chloroplast proteins was examined to determine how many proteins were synthesized inside the chloroplast. Cells were labeled in vivo with [35S]-sulfate in the presence of either. chloroplast or cytoplasmic protein synthesis inhibitors, and fractionated into soluble proteins, thylakoid membranes, and chloroplast ribosomal subunits. The use of high specific activity sulfate and improved gel electrophoretic techniques were key features of this study because they permitted minor chloroplast products to be detected for the first time. It was determined that twenty-eight to thirty-one polypeptides are synthesized in the chloroplast. These include the major thylakoid membrane proteins previously reported to be chloroplast products by Chua and Gillham (1977), but also include a number of minor and low-molecular weight thylakoid membrane proteins, three soluble proteins, and at least four chloroplast ribosomal proteins. The synthesis of chloroplast envelope constituents was not examined. To find out which thylakoid membrane proteins were integral and which peripheral, membranes were extracted with acid, base, urea, and guanidine hydrochloride. It was determined that both integral and peripheral membrane proteins are synthesized in the chloroplast. Integral proteins included constituent polypeptides of chlorophyll-protein complexes and polypeptide D1; the most notable peripheral proteins synthesized in the chloroplast were polypeptides believed to be the α and β coupling factor subunits. In an effort to gain information about the biosynthetic pathway followed by chloroplast synthesized polypeptides, poly(A)- mRNA was isolated and translated in the wheat germ cell-free system. One product was identified as the ribulose-1,5-bisphosphate carboxylase large subunit and was synthesized in vitro in a form indistinguishable from the authentic polypeptide. A second product was tentatively identified as the integral membrane protein D1. The in vitro synthesized polypeptide was slightly different from authentic D1 in a number of ways. The relationship of these differences to a possible precursor in vivo is discussed.
License and Reuse Information
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Recommended Citation
Matlin, Karl Stanley, "Studies on Chloroplast Protein Synthesis In Vivo and In Vitro" (1979). Student Theses and Dissertations. 532.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/532
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy