QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kerscher, S. J. Articles by Brandt, U. Search for Related Content PubMed PubMed Citation Articles by Kerscher, S. J. Articles by Brandt, U.

External alternative NADH:ubiquinone oxidoreductase redirected to the internal face of the mitochondrial inner membrane rescues complex I deficiency in Yarrowia lipolytica

Stefan J. Kerscher^1,, Andrea Eschemann¹, Pamela M. Okun^1,* and Ulrich Brandt¹

¹ Universitätsklinikum Frankfurt, Institut für Biochemie I, D-60590 Frankfurt am Main, Federal Republic of Germany
^* Present address: Universität Heidelberg, Institut für Molekulare Genetik, D-69120 Heidelberg, Federal Republic of Germany

View larger version (11K): [in a new window]   Fig. 1. Strategy for the deletion of the Y. lipolytica NUAM gene encoding the 75 kDa subunit of complex I. (A) Construction of the nuam::URA3 deletion allele. (B) Deletion of one of the genomic NUAM copies in diploid GB1 cells by homologous recombination.

View larger version (16K): [in a new window]   Fig. 2. Plasmid pUB22 for expression of the internal version of the alternative NADH:ubiquinone oxidoreductase of Y. lipolytica. Insert sequences derived from the NDH2 gene locus are depicted in black, sequences derived from the NUAM gene locus are depicted in gray. Unique restriction sites are indicated.

View larger version (27K): [in a new window]   Fig. 3. Internal expression of alternative NADH:ubiquinone oxidoreductase allows survival in the presence of the complex I inhibitor DQA and survival of nuam cells. See text for details.

View larger version (23K): [in a new window]   Fig. 4. DQA resistance conferred by the YLNUAM-ECNDH gene fusion. The gene dosage effect is evident from comparing the phenotypes of ndh2 pUB10 (one gene copy) and ndh2 pUB11 (two gene copies) cells. The phenotype of ndh2 pUB22 cells is included for comparison.

View larger version (26K): [in a new window]   Fig. 5. SCNDI1 cannot be imported into Y. lipolytica mitochondria. On a western blot of intact mitochondria (1.5 µg of total protein) from S. cerevisiae KM91 (S.c.) and from Y. lipolytica ndh2::URA3, pUB4::SCNDI1 (Y.l.) probed with -SCNDI1 monoclonal antibody 22-2-B8, SCNDI1 is not detectable in Y. lipolytica.

View larger version (15K): [in a new window]   Fig. 6. SCNDI1 does not confer DQA resistance, either with the wild-type -1 arginine (pUB28) or the genetically engineered -3 arginine (pUB27) processing site.