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Title: THE EXPRESSION AND ASSEMBLY OF THE E1 COMPONENT OF MITOCHONDRIAL PYRUVATE DEHYDROGENASE COMPLEX OF PISUM SATIVUM IN THE CYTOPLASM OF THE METHYLOTROPHIC YEAST PICHIA PASTORIS

Author
item MORENO, J - U OF MO, COLUMBIA, MO
item Miernyk, Jan
item RANDALL, D - U OF MO, COLUMBIA, MO

Submitted to: American Society of Plant Physiologists Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 7/16/1999
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: The mitochondrial pyruvate dehydrogenase complex (mtPDC) catalyzes the oxidative decarboxilation of pyruvate to acetyl-CoA, CO2, and NADH, thus linking glycolysis to the Krebs cycle. The mtPDC has three primary components, E1, E2, and E3 that act sequentially. In addition, a PDH kinase and a P-PDH phosphatase are also associated with this complex to regulate the mtPDC by reversible phosphorylation. The E1 component is composed of and subunits to form a 2 2 heterotetramer, which catalyzes the first step of pyruvate decarboxylation. We have cloned the cDNA for both E1 subunits from Pisum sativum. In order to fully establish the regulation of the mtPDC, it is essential to have an expression system that provides functional E1. We have selected the methylotrophic yeast Pichia pastoris for the cytoplasmic expression of both subunits of the pea mtE1. Both subunits were put under the control of the AOX1 promoter (inducible by methanol) and the E1 harbored a poly-His affinity tag at the C-terminus while E1 did not. Both subunits could be expressed individually in soluble form or they could be co-expressed in soluble form. When both subunits were co-expressed they could be co-immunoprecipitated with antibodies to E1 and co-purified by affinity chromatography suggesting that they were associated. The E1/E1 interaction was resistant to vigorous washing with 0.5% Triton X-100, 800 mM KCl, and 10 mM imidazole. Clarified cell homogenates from co-expressing recombinant strain exhibited 2-fold greater pyruvate decarboxilation activity than control homogenates. Pichia pastoris provides an excellent system for the expression of recombinant plant pyruvate dehydrogenase. This research supported by NSF and the MoAES and Food for 21st Century Programs.