|Mooney, B - UNIV OF MISSOURI-COLUMBIA|
|Randall, D - UNIV OF MISSOURI-COLUMBIA|
Submitted to: American Society of Plant Biologists Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: July 3, 2001
Publication Date: N/A
Technical Abstract: The dihydrolipoyl acetyltransferase (E2) component forms the 60-subunit core of the pyruvate dehydrogenase complex. To determine the regions of Arabidopsis thaliana mitochondrial E2 important in assembly, portions of E2 were co-expressed in E. coli with six histidine (His6) or glutathione S- transferase (GST) affinity tags. When glutathione-Sepharose was used to capture the GST-fusion proteins, His6 proteins co-purified demonstrating protein association. Association was specific for E2, independent of the GST moiety, and only N- and C-terminal portions of the catalytic domain associated. Full-length E2 was expressed in E. coli and formed a high molecular mass complex of 60 subunits, with icosahedral symmetry, as determined by size-exclusion chromatography and electron microscopy, respectively. Deletion of the last three highly-conserved amino acids did not affect assembly of the 60mer, while deletion of the last four abolished d60mer formation, but had no effect upon trimer formation. The C-terminal four amino acid deletion disrupted helical secondary structure at the extreme C-terminus. Similarly, site-directed mutagensis of R634 causing disruption of the helical structure also abolished 60mer, formation but not trimer formation. These data show that the N-terminal region of the catalytic domain is required for trimer assembly while C-terminal helical secondary structure is required for inter-trimer association and concomitant 60mer formation.