UC San Diego

Chloroplasts house the photosynthetic apparatus that allows oxygenic photosynthesis to occur. The assembly of these core complexes requires both proteins that are synthesized in the plastid as well as proteins that are translocated across the chloroplast envelope from the cytosol. In both instances, the photosynthetic subunits need to be folded or re-folded into the correct confirmation to ensure their functionality. To assist with protein folding, chloroplasts have evolved to contain a large number of molecular chaperones that prevent protein polypeptide chains from aggregating before they can achieve their native state. In addition to molecular chaperones, chloroplasts have also evolved to contain a protein disulfide isomerase that is involved in redox signaling and which can potentially be used to form disulfide bridges in complex multi-subunit proteins. Although some of the chloroplast protein folding machinery is of a eukaryotic origin most the components still resemble those of their prokaryotic ancestors. To increase our understanding of the complex protein folding machinery of chloroplasts we examined the ability of Chlamydomonas reinhardtii chloroplasts to fold and assemble complex multi-subunit proteins such as a full-length human antibody and immuotoxin proteins. The assembly of a human antibody is a complex process that requires molecular chaperones to hold proteins in a non-aggregated state while a protein disulfide isomerase catalyzes the formation of 16 disulfide bonds. The ability to assemble an antibody demonstrates biochemically that the protein disulfide isomerase in chloroplast is capable of functioning cohesively with the plastid molecular chaperones to form disulfide bridges. We were also able to produce enzymatically active immuntoxin proteins in chloroplasts and demonstrate that they were able to target and kill specific cancer cells. This ability to accumulate immunotoxin proteins demonstrates that chloroplast protein transport is unidirectional and that no proteins escape the chloroplast. Many of the biological protein folding components have been identified in chloroplasts but little is known about how they function cohesively to allow complex proteins to fold into their native states. A thorough understanding of the biochemical nature of chloroplast protein folding and how they function together will supplement the basic knowledge of protein folding in all organisms and may also assist in the design and use of algae as a biotechnological tool to generate difficult to produce multi-subunit proteins