Date of Award

1988

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Sue G. Bartlett

Abstract

The small subunits of the chloroplast enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) are synthesized in the cytoplasm as precursors and transported into the organelle. The precursor (pS) of the small subunit (S) of RuBisCO contains an amino-terminal extension, the transit peptide, which is removed during or after transport into the chloroplast. The possible involvement of an RNA-containing component in transport of proteins into chloroplasts was investigated using a reconstituted system of pS translated in vitro and isolated chloroplasts. The post-ribosomal supernatant of the in vitro translation mixture and/or isolated chloroplasts were treated with ribonuclease prior to incubation for transport. Transport of pS was unaffected by the ribonuclease treatment indicating that an RNA is not involved in this process. While the targeting function of the pS transit peptide is well documented, the role in transport of the mature portion of the precursor has not been elucidated. Mutations in the mature portion of pS were constructed to assess the influence of the mature portion of the precursor on transport. Each mutant pS was analysed for transport into chloroplasts and assembly into RuBisCO. Mutations in the amino-terminal region of S severely interfere with transport of pS while mutations near the middle of S interfere to a lesser degree. These results indicate that the pS transit peptide is tailored for optimal transport of S. Assembly of mutant S into holoenzyme was poor, and a significant fraction was associated with RuBisCO binding protein which is postulated to assist assembly of the holoenzyme. Mutant S may be arrested at an intermediate step in the pathway of assembly. RuBisCO binding protein is homologous to the Escherichia coli groEL protein which binds unfolded proteins and mediates assembly of protein complexes. Fusion proteins containing pS or S fused to the carboxy-terminus of Staphylococcus protein A were expressed in E. coli and purified by protein A-affinity chromatography. GroEL protein copurified with protein A-pS and protein A-S but not protein A alone. Association of groEL protein with S expressed in E. coli indicates that RuBisCO binding protein, like groEL protein, may bind other unfolded or unassembled chloroplast proteins in addition to S.

Pages

113

DOI

10.31390/gradschool_disstheses.4654

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