EXPOSED THIOLS CONFER LOCALIZATION IN THE ENDOPLASMIC-RETICULUM BY RETENTION RATHER THAN RETRIEVAL

The cysteine present in the Ig mu chain tailpiece (mu tp) prevents the secretion of unpolymerized IgM intermediates and causes their accumul ation in the endoplasmic reticulum (ER), In principle, this can be the consequence of actual retention in this organelle or of retrieval fro m the Gels. To determine which of the two mechanisms underlies the cys teine dependent ER localization, we analyze here the post-translationa l modifications of suitably engineered cathepsin D (CD) molecules, The glycans of this protease are phosphorylated by post-ER phosphotransfe rases and further modified in the trans-Golgi to generate a mannose 6- phosphate lysosome targeting signal, Only trace amounts of the mu tp-t agged CD (CDM mu tpCys) are phosphorylated, unless retention is revers ed by exogenous reducing agents or the critical cysteine mutated (CDM mu tpSer), In contrast, a KDEL-tagged CD, that is retrieved from the G els into the ER, acquires phosphates, though mainly resistant to alkal ine phosphatase. Similarly to CDM mu tpSer, the few CDM mu tpCys molec ules that escape retention and acquire phosphates in the cis-Golgi are transported beyond the KDEL retrieval compartment, as indicated by th eir sensitivity to alkaline phosphatase. These results demonstrate tha t the thiol-dependent ER localization arises primarily from true reten tion, without recycling through the Golgi.