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Volume 271, Number 41, Issue of October 11, 1996 pp. 25208-25212
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Covalent Attachment of FAD Derivatives to a Fusion Protein Consisting of 6-Hydroxy-D-nicotine Oxidase and a Mitochondrial Presequence
FOLDING, ENZYME ACTIVITY, AND IMPORT OF THE MODIFIED PROTEIN INTO YEAST MITOCHONDRIA

(Received for publication, June 18, 1996)

Michaela Stoltz , Joachim Rassow , Andreas F. Bückmann and Roderich Brandsch

From the Institut für Biochemie und Molekularbiologie, D-79104 Freiburg, Germany and the  Gesellschaft für Biotechnologische Forschung, D-38124 Braunschweig, Germany

Autoflavinylation of 6-hydroxy-D-nicotine oxidase (6-HDNO) was successfully employed to modify the protein covalently with FAD derivatives. The model compounds N⁶-(2-aminoethyl)-FAD and N⁶-(6-carboxyhexyl)-FAD were spontaneously bound to a fusion protein consisting of the mitochondrial targeting sequence of Neurospora crassa F₀-ATPase subunit 9 (Su9) attached to 6-HDNO. When translated in the rabbit reticulocyte lysate, Su9-6-HDNO was in the trypsin-sensitive apoenzyme form; when translated in the presence of flavins it adopted a trypsin-resistant conformation characteristic of the 6-HDNO holoenzyme. With flavin derivatives, Su9-6-HDNO exhibited approximately 50% of the 6-HDNO activity observed with FAD.

The covalently modified Su9-6-HDNO was imported into Saccharomyces cerevisiae mitochondria with an efficiency equal to that of the apoenzyme. Apparently the increase in size and charge of the FAD moiety did not hamper translocation across the mitochondrial membranes. Yeast mutant ssc1-2 mitochondria deficient in mtHsp70 unfoldase activity imported the flavinylated Su9-6-HDNO protein. In mutant ssc1-3 mitochondria deficient in both mtHsp70 unfoldase and translocase activity Su9-6-HDNO was trapped as translocation intermediate; the Su9 presequence was passed to the matrix where it was proteolytically cleaved by the mitochondrial processing peptidase; (MPP); the translocation-arrested 6-HDNO moiety adopted a trypsin-sensitive conformation. Our results indicate that unfolding of the FAD-stabilized flavin-binding domain of 6-HDNO in passage through the mitochondrial general insertion pore does not require the activity of mtHsp70.

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