Date of Award


Document Type

Dissertation - Restricted

Degree Name

Doctor of Philosophy (PhD)


Biological Sciences

First Advisor

Friedman, Alan

Second Advisor

Kumaran, Krishna

Third Advisor

Fredricks, Walter W.


Chloroplasts play a central role in photosynthesis and many important aspects of plant cellular metabolism. Although chloroplasts contain an autonomous genome, they synthesize only a small subset of the proteins required for photosynthesis and other processes that occur within the organelle. The vast majority of the proteins in the chloroplast are encoded in the nucleus and synthesized on free ribosomes in the cytoplasm as higher molecular weight precursors that posttranslationally import into chloroplasts. Although a wealth of information is known about protein import into chloroplasts, the actual mechanism by which proteins translocate across the outer and inner chloroplast envelope membranes remains obscure. In this study, a series chimeric fusion proteins between the Rubisco small subunit precursor (prSS) and mouse cytosolic dihydrofolic reductase (DHFR) were constructed and expressed. The prSS-DHFR fusion proteins were expressed in a wheat germ extract. Upon expression, the dihydrofolate reductase domains of these fusion proteins remained protease sensitive in the presence of methotrexate and failed to bind to methotrexate column. These data demonstrate that the DHFR domain of the fusion proteins folded into a non-native conformation. However, the DHFR domains of the prSS-DHFR fusion proteins folded into a native, protease resistant, conformation when translated in the wheat germ extract containing the DHFR ligands (either methotrexte, NADPH, or dihydrofolate). The import of the prSS-DHFR fusion proteins was examined in an in vitro chloroplast import assay. Methotrexate did not prevent the import of the DHFR fusion proteins into chloroplast. However, it was demonstrated in this dissertation that the DHFR fusion protein must unfold druing chloroplast protein import and refold within the organelle. These results support a vectorial model of chloroplast protein import.



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