Characterization of posttranslational formylglycine formation by luminal components of the endoplasmic reticulum.
C(alpha)-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 that allowed us 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/mol, and the pH optimum is approximately 10. The activity is sensitive to the SH/SS equilibrium and is stimulated by Ca(2+). Formylglycine formation is efficiently inhibited by a synthetic sulfatase peptide representing the sequence directing formylglycine modification. The established assay system should make possible the biochemical identification of the modifying enzyme.
Fey
J
J
Institut für Biochemie und Molekulare Zellbiologie, Abt. Biochemie II, Universität Göttingen, Heinrich-Düker-Weg 12, Göttingen 37073, Germany.
Balleininger
M
M
Borissenko
L V
LV
Schmidt
B
B
von Figura
K
K
Dierks
T
T
D000409:Alanine / Q000031:analogs & derivatives* / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002417:Cattle; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005998:Glycine / Q000031:analogs & derivatives* / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010179:Pancreas / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D014176:Protein Biosynthesis; D011497:Protein O-Methyltransferase / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D012270:Ribosomes / Q000378:metabolism; D012492:Salts / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D003902:Detergents; D010455:Peptides; D012492:Salts; C524739:C(alpha)-formylglycine; D011497:Protein O-Methyltransferase; D000409:Alanine; D002118:Calcium; D005998:Glycine