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M108943200v1
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Papers In Press, published online ahead of print October 12, 2001
J. Biol. Chem, 10.1074/jbc.M108943200
Submitted on September 17, 2001
Revised on October 12, 2001
Accepted on October 12, 2001

Characterization of posttranslational formylglycine formation by lumenal components of the endoplasmic reticulum

Jens Fey, Martina Balleininger, Ljudmila V. Borissenko, Bernhard Schmidt, Kurt von Figura, and Thomas Dierks

Biochemistry 2, University of Goettingen, Goettingen D-37073

Corresponding Author: tdierks{at}gwdg.de

Calpha -formylglycine is the key catalytic residue in the active site of sulfatases. In eukaryotes formylglycine is generated during or immediately after sulfatase translocation into the endoplasmic reticulum by oxidation of a specific cysteine residue. We established an in vitro assay allowing to measure formylglycine modification independent of protein translocation. The modifying enzyme was recovered in a microsomal detergent extract. As a substrate we used ribosome-associated nascent chain complexes comprising in vitro synthesized sulfatase fragments that were released from the ribosomes by puromycin. Formylglycine modification was highly efficient and did not require a signal sequence in the substrate polypeptide. Ribosome-association helped to maintain the modification competence of nascent chains, but only after their release efficient modification occurred. The modifying machinery consists of soluble components of the endoplasmic reticulum lumen, as shown by differential extraction of microsomes. The in vitro assay can be performed under kinetically controlled conditions. The activation energy for formylglycine formation is 61 kJ/mole, the pH optimum approximately 10. The activity is sensitive to the -SH/-SS-equilibrium and is stimulated by Ca2+. Formylglycine formation is efficiently inhibited by a synthetic sulfatase peptide representing the sequence directing formylglycine modification. The established assay system should allow to biochemically identify the modifying enzyme.


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