The cysteine present in the Ig μ chain tailpiece (μtp) 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 μtp-tagged CD (CDMμtpCys) are phosphorylated, unless retention is reversed by exogenous reducing agents or the critical cysteine mutated (CDMμtpSer). 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 CDMμtpSer, the few CDMμtpCys 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.
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