Protein Targeting to Endoplasmic Reticulum by Dilysine Signals Involves Direct Retention in Addition to Retrieval*
Abstract
Dilysine signals confer localization of type I membrane proteins to the endoplasmic reticulum (ER). According to the prevailing model these signals target proteins to the ER by COP I-mediated retrieval from post-ER compartments, whereas the actual retention mechanism in the ER is unknown. We expressed chimeric membrane proteins with a C-terminal -Lys-Lys-Ala-Ala (KKAA) or -Lys-Lys-Phe-Phe (KKFF) dilysine signal in Lec-1 cells. Unlike KKFF constructs, which had access to post-ER compartments, the KKAA chimeras were localized to the ER by confocal microscopy and were neither processed by cis-Golgi-specific enzymes in vivonor included into ER-derived transport vesicles in an in vitro budding assay, suggesting that KKAA-bearing proteins are permanently retained in the ER. The ER localization was nonsaturable and exclusively mediated by the dilysine signal because mutating the lysines to alanines led to cell surface expression of the chimeras. Although the KKAA signal avidly binds COP I in vitro, the ER retention by this signal does not depend on intact COP I in vivo because it was not affected in an ε-COP-deficient cell line. We propose that dilysine ER targeting signals can mediate ER retention in addition to retrieval.
Footnotes
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↵* This work was supported by grants from the Swiss National Science Foundation and the Kantons of Basel and an EMBO long term fellowship (to H. A.).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.
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↵‡ To whom correspondence should be addressed: Dept. of Pharmacology, Biozentrum, University of Basel, Klingelbergstr. 70, CH-4056 Basel, Switzerland. Tel.: 41-61-267-22-22; Fax: 41-61-267-22-08; E-mail:Hauri{at}ubaclu.unibas.ch.
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↵2 M. Gomez, personal communication.
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↵3 H. Andersson and H.-P. Hauri, unpublished observations.
- Abbreviations:
- ER
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endoplasmic reticulum
- endo-D
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endoglycosidase D
- ERGIC
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ER/Golgi intermediate compartment
- mAb
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monoclonal antibody
- PAGE
-
polyacrylamide gel electrophoresis
- HSP
-
high speed pellet
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- Received January 27, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
