title: Exposed thiols confer localization in the endoplasmic reticulum by retention rather than retrieval.
abstract: The cysteine present in the Ig micro chain tailpiece (microtp) prevents the secretion of unpolymerized IgM intermediates and causes their accumulation in the endoplasmic reticulum (ER). In principle, this can be the consequence of actual retention in this organelle or of retrieval from the Golgi. To determine which of the two mechanisms underlies the cysteine-dependent ER localization, we analyze here the post-translational modifications of suitably engineered cathepsin D (CD) molecules. The glycans of this protease are phosphorylated by post-ER phosphotransferases and further modified in the trans-Golgi to generate a mannose 6-phosphate lysosome targeting signal. Only trace amounts of the mutp-tagged CD (CDM&mutpCys) are phosphorylated, unless retention is reversed by exogenous reducing agents or the critical cysteine mutated (CDMmutpSer). In contrast, a KDEL-tagged CD, that is retrieved from the Golgi into the ER, acquires phosphates, though mainly resistant to alkaline phosphatase. Similarly to CDMmutpSer, the few CDMmutpCys molecules that escape retention and acquire phosphates in the cis-Golgi are transported beyond the KDEL retrieval compartment, as indicated by their sensitivity to alkaline phosphatase. These results demonstrate that the thiol-dependent ER localization arises primarily from true retention, without recycling through the Golgi.
authors.affiliation: Dipartimento di Medicina ed Oncologia Sperimentale, Sez. di Patologia Generale, Università di Torino, 10125 Torino, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Isidoro
authors.forename: C
authors.initials: C
authors.lastname: Maggioni
authors.forename: M
authors.initials: M
authors.lastname: Demoz
authors.forename: A
authors.initials: A
authors.lastname: Pizzagalli
authors.forename: A M
authors.initials: AM
authors.lastname: Fra
authors.forename: R
authors.initials: R
authors.lastname: Sitia
mesh_terms: D000818:Animals; D001692:Biological Transport; D019556:COS Cells; D002402:Cathepsin D / Q000235:genetics / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D000882:Haplorhini; D007075:Immunoglobulin M / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D009842:Oligopeptides / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014162:Transfection
chemical_list: D007075:Immunoglobulin M; D009842:Oligopeptides; D010710:Phosphates; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013438:Sulfhydryl Compounds; C073762:lysyl-aspartyl-glutamyl-leucine; D002402:Cathepsin D