O-Glycosylation of a Secretory Granule Membrane Enzyme Is Essential for Its Endocytic Trafficking
| Autor: | Betty A. Eipper, TuKiet T. Lam, Nils Bäck, Richard E. Mains, Kurutihalli S. Vishwanatha |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Glycosylation Endocytic cycle Biological Transport Active Biology Biochemistry Mixed Function Oxygenases 03 medical and health sciences chemistry.chemical_compound stomatognathic system Multienzyme Complexes Cell Line Tumor parasitic diseases Animals Molecular Biology Furin Secretory granule membrane reproductive and urinary physiology Secretory Vesicles Cell Membrane Cell Biology Lyase Endocytosis Rats Cytosol 030104 developmental biology Secretory protein Peptide amidation chemistry embryonic structures biology.protein |
| Zdroj: | Journal of Biological Chemistry. 291:9835-9850 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m115.711838 |
| Popis: | Peptidylglycine α-amidating monooxygenase (PAM) (EC 1.14.17.3) catalyzes peptide amidation, a crucial post-translational modification, through the sequential actions of its monooxygenase (peptidylglycine α-hydroxylating monooxygenase) and lyase (peptidyl-α-hydroxyglycine α-amidating lyase (PAL)) domains. Alternative splicing generates two different regions that connect the protease-resistant catalytic domains. Inclusion of exon 16 introduces a pair of Lys residues, providing a site for controlled endoproteolytic cleavage of PAM and the separation of soluble peptidylglycine α-hydroxylating monooxygenase from membrane-associated PAL. Exon 16 also includes two O-glycosylation sites. PAM-1 lacking both glycosylation sites (PAM-1/OSX; where OSX is O-glycan-depleted mutant of PAM-1) was stably expressed in AtT-20 corticotrope tumor cells. In PAM-1/OSX, a cleavage site for furin-like convertases was exposed, generating a shorter form of membrane-associated PAL. The endocytic trafficking of PAM-1/OSX differed dramatically from that of PAM-1. A soluble fragment of the cytosolic domain of PAM-1 was produced in the endocytic pathway and entered the nucleus; very little soluble fragment of the cytosolic domain was produced from PAM-1/OSX. Internalized PAM-1/OSX was rapidly degraded; unlike PAM-1, very little internalized PAM-1/OSX was detected in multivesicular bodies. Blue native PAGE analysis identified high molecular weight complexes containing PAM-1; the ability of PAM-1/OSX to form similar complexes was markedly diminished. By promoting the formation of high molecular weight complexes, O-glycans may facilitate the recycling of PAM-1 through the endocytic compartment. |
| Databáze: | OpenAIRE |
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