Abstract
Inside the endoplasmic reticulum (ER) formylglycine-generating enzyme (FGE) catalyzes in newly synthesized sulfatases the post-translational oxidation of a specific cysteine. Thereby formylglycine is generated, which is essential for sulfatase activity. Here we show that ERp44 interacts with FGE forming heterodimeric and, to a lesser extent, also heterotetrameric and octameric complexes, which are stabilized through disulfide bonding between cysteine 29 of ERp44 and cysteines 50 and 52 in the N-terminal region of FGE. ERp44 mediates FGE retrieval to the ER via its C-terminal RDEL signal. Increasing ERp44 levels by overexpression enhances and decreasing ERp44 levels by silencing reduces ER retention of FGE. Suppressing disulfide bonding by mutating the critical cysteines neither abrogates ERp44.FGE complex formation nor interferes with ERp44-mediated retention of FGE, indicating that noncovalent interactions between ERp44 and FGE are sufficient to mediate ER retention. The N-terminal region of FGE harboring Cys(50) and Cys(52) is dispensible for catalytic activity in vitro but required for FGE-mediated activation of sulfatases in vivo. This in vivo activity is affected neither by overexpression nor by silencing of ERp44, indicating that a further ER component interacting with the N-terminal extension of FGE is critical for sulfatase activation.
Highlights
The structural similarity of paralog of formylglycine-generating enzyme (FGE) (pFGE) to FGE enables the FGE1– 88-pFGE fusion described above to present the N-terminal extension to its catalytic FGE partner and thereby to complement in vivo functionality, i.e. FGly generation in nascent sulfatase polypeptides inside the endoplasmic reticulum (ER).7
We could further show that a Cys-Gly-Cys motif in this N-terminal extension of FGE, which is fully conserved in all known eukaryotic FGE sequences, is critical for activation of sulfatases, whereas it is dispensible for ER retention of FGE.7
The complexes are stabilized by disulfide bridges involving Cys50 and Cys52 in the N-terminal extension of FGE and Cys29 in ERp44
Summary
In eukaryotes newly synthesized sulfatase polypeptides undergo FGly modification in the lumen of the endoplasmic reticulum (ER) by late co- or early post-translational oxidation of a critical cysteine residue that is part of a highly conserved consensus motif (C(T/S/C/A)PSR for human sulfatases) [13, 14] The oxidation of this cysteine is catalyzed by the recently discovered FGly-generating enzyme (FGE) [15, 16] through a novel mixed-functional oxygenase mechanism [17, 18].5. We could further show that a Cys-Gly-Cys motif in this N-terminal extension of FGE, which is fully conserved in all known eukaryotic FGE sequences, is critical for activation of sulfatases, whereas it is dispensible for ER retention of FGE.7 This led us to postulate that the N-terminal extension of FGE mediates the interaction with ER components, which are required for the generation of FGly residues and the retention of FGE in the ER. In this study we describe the identification of an ER component interacting with the N-terminal extension of FGE, the biochemical basis of this interaction, and its direct functional relevance for FGE retention in the ER
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