Exposed Thiols Confer Localization in the Endoplasmic Reticulum by Retention Rather than Retrieval

doi:10.1074/jbc.271.42.26138

Exposed Thiols Confer Localization in the Endoplasmic Reticulum by Retention Rather than Retrieval*

From the ^‡ Dipartimento di Medicina ed Oncologia Sperimentale, Sez. di Patologia Generale, Università di Torino, 10125 Torino and the
^¶ DIBIT-San Raffaele Scientific Institute, 20132 Milano, Italy

^∥ To whom correspondence should be addressed:
DIBIT, San Raffaele Scientific Inst., via Olgettina 58, 20132 Milano, Italy.
Tel.: 39-2-2643-4722; Fax: 39-2-2643-4723; E-mail: sitiar{at}dibit.hsr.it

Abstract

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.

Footnotes

↵^§ These authors contributed equally to this work.
↵* This work was supported in part through grants from Ministero della Sanità (AIDS Special Project), Associazione Italiana per la Ricerca sul Cancro and Consiglio Nazionale delle Ricerche (Progretti Finalizzati ACRO, BBTS, and IG). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

ER

endoplasmic reticulum

AP

alkaline phosphatase

CD

cathepsin D

CDM

Myc-tagged CD

CDMK

CDM + KDEL

CDMμtpCys

CDM + the Ig μ tailpiece (Cys⁵⁷⁵)

CDMμtpSer

CDM + the Ig μ tailpiece (Ser⁵⁷⁵)

Ig

immunoglobulin

2-ME

2-mercaptoethanol

Endo-H

endo-β-N-acetylglucosaminidase H

PAGE

polyacrylamide gel electrophoresis.
↵² C. Maggioni, C. Isidoro, A. Fra, and R. Sitia, unpublished data.
- Received May 30, 1996.
- Revision received July 11, 1996.