Abstract
We analyzed the folding, covalent flavinylation, and mitochondrial import of the rabbit reticulocyte lysate-translated bacterial 6-hydroxy-D-nicotine oxidase (6-HDNO) fused to the mitochondrial targeting sequence of rat liver dimethylglycine dehydrogenase. Translation of 6-HDNO in FAD-supplemented reticulocyte lysate resulted in a protein that contained covalently incorporated FAD, exhibited enzyme activity, and was trypsin-resistant, a characteristic of the tight conformation of the holoenzyme. The attached mitochondrial presequence did not prevent folding, binding of FAD, or enzyme activity of the 6-HDNO moiety of the fusion protein (pre-6-HDNO). Pre-6-HDNO was imported into rat liver mitochondria and processed by the mitochondrial processing peptidase. Incubation of the trypsin-resistant pre-holo-6-HDNO protein with deenergized rat liver mitochondria demonstrated that upon contact with mitochondria, the protein was unfolded and became trypsin sensitive. Mitochondrial import assays showed that the unfolded pre-holo-6-HDNO with covalently attached FAD was imported into rat liver mitochondria. Inside the mitochondrion the holo-6-HDNO was refolded into the trypsin-resistant conformation. However, when pre-apo-6-HDNO was imported only part of the protein became trypsin resistant (approximately 20%). Addition of FAD and the allosteric effector glycerol 3-phosphate to apo-6-HDNO containing mitochondrial matrix was required to transform the protein into the trypsin-resistant conformation characteristic of holo-6-HDNO.
Highlights
From the Biochemisches Institut, Uniuersitat Freiburg, Freiburg, Federal Republic of Germany and the Wepartment of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510
We show that 1) the mitochondrial targeting sequence does not prevent folding, flavinylation, or enzyme activity of the fusion protein synthesized in the reticulocyte lysate (RL); 2) the fusion protein is imported into rat liver mitochondria and correctly processed; 3) the compact, trypsinresistant conformation of the flavinylated precursor is unfolded upon interaction with the mitochondrion; and 4) flavinylation of the precursor does not prevent its import
Holoenzyme Formation, and [14C1FAD Incorporation into 6-hydroxy-D-nicotine oxidase (6-HDNO) and Pre-6-HDNO Translated in the in Vitro RL System-Since the autocatalytic incorporation of FAD into 6-HDNO depends on the folding of the polypeptide into a flavinylation-competent conformation [10], we asked whether 6-HDNO translated in the RL folds correctly and exhibits cofactor attachment and enzyme activity
Summary
From the Biochemisches Institut, Uniuersitat Freiburg, Freiburg, Federal Republic of Germany and the Wepartment of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510. Mitochondrial import assays showed that the unfolded pre-holo-6-HDNO with covalently attached FAD was imported into rat liver mitochondria. Import of nuclear encoded precursor proteins of mitochondrially located enzymes seems to depend on a loosely folded, import-competent conformation of the polypeptide (for a review, see Ref. 1). Binding of the cofactor in several enzymes takes place late in the folding pathway, when the protein has reached a native-like conformation, and results in the stabilization of the native structure of the holoenzyme These findings raise the question whether precursor proteins with bound cofactor are imported into mitochondria
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