Date of Award

8-2009

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Barry D. Bruce

Committee Members

Gladys Alexandre, Beth C. Mullin

Abstract

The majority of chloroplast proteins are nuclear encoded and transcribed on cytosolic ribosomes, and therefore must be post-translationally imported into the chloroplast. Preproteins are directed to the chloroplast via a cleavable Nterminal extension known as a transit peptide. This transport is mediated by the Toc and Tic complexes (Translocon at the Outer/Inner Chloroplast envelope membrane), functioning in tandem to transport preproteins into chloroplasts relying on the hydrolysis of ATP and GTP. The Toc complex is composed of the β-barrel channel protein Toc75 and the homologous GTPase receptors Toc34 and Toc159. GTP hydrolysis is necessary for the formation of the early import intermediate, in which the transit peptide is inserted into the Toc channel, but the presence of internal ATP is the only energetic requirement for the later stages of translocation to occur, mediated by the stromal motor complex with an Hsp100 isoform hydrolyzing stromal ATP. The purpose of the current study is to characterize the change in stability and/or oligomeric status of the Toc complex with the incubation of nucleotides, analogs, proteins/peptides, etc. by blue native electrophoresis followed by 2d SDS-PAGE. The Toc complex ranges from ~800 kDa to greater than 1320 kDa for the proposed Toc/Tic supercomplex when no proteolytic degradation has occurred. Proteolytic degradation of Toc159 is correlated with the appearance of complexes with a mass ranging from 800 kDa to 440 kDa and below. Proteolytic degradation of Toc159 is more apparent in chloroplasts purified from older Pisum sativum plants. The results of the incubation of chloroplasts with GDP, GTP, and non-hydrolyzable analogs before analysis by 2d electrophoresis followed by western blot hybridization suggest that the loading of the GTPase receptors with nucleotide triphosphate results in the increased association of Toc components in complexes in the size range of 880- 630 kDa.

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