Efficient large scale purification of his-tagged proton translocating NADH: ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica

Proton translocating NADH:ubiquinone oxidoreductase (complex I) is the larg est membrane bound multiprotein complex of the respiratory chain and the on ly one for which no molecular structure is available so far. Thus, informat ion on the mechanism of this central enzyme of aerobic energy metabolism is still very limited. As a new approach to analyze complex I, we have recent ly established the strictly aerobic yeast Yarrowia lipolytica as a model sy stem that offers a complete set of convenient genetic tools and contains a complex I that is stable after isolation. For crystallization of complex I and to obtain its molecular structure it is a prerequisite to prepare large amounts of highly pure enzyme. Here we present the construction of his-tag ged complex I that for the first time allows efficient affinity purificatio n. Our protocol recovers almost 40% of complex I present in Yarrowia mitoch ondrial membranes. Overall, 40-80 mg highly pure and homogeneous complex I can be obtained from 10 1 of an overnight Y. lipolytica culture. After reco nstitution into asolectin proteoliposomes, the purified enzyme exhibits ful l NADH:ubiquinone oxidoreductase activity, is fully sensitive to inhibition by quinone analogue inhibitors and capable of generating a proton-motive f orce. (C) 2001 Elsevier Science B.V. All rights reserved.