A Casein Kinase II Phosphorylation Site in the Cytoplasmic Domain of the Cation-dependent Mannose 6-Phosphate Receptor Determines the High Affinity Interaction of the AP-1 Golgi Assembly Proteins with Membranes (*)
- From the European Molecular Biology Laboratory, Postfach 10-2209, Meyerhofstrasse 1, D-69012 Heidelberg, Federal Republic of Germany
- ¶ To whom correspondence should be addressed. Tel.: 49-6221-387-285; Fax: 49-6221-387-306.
Abstract
The transport of proteins from the secretory to the endocytic pathway is mediated by carrier vesicles coated with the AP-1 Golgi assembly proteins and clathrin. The mannose 6-phosphate receptors (MPRs) are two major transmembrane proteins segregated into these transport vesicles. Together with the GTPase ARF-1, these cargo proteins are essential components for the efficient translocation of the cytosolic AP-1 onto membranes of the trans-Golgi network, the first step of clathrin coat assembly. MPR-negative fibroblasts have a low capacity of recruiting AP-1 which can be restored by re-expressing the MPRs in these cells. This property was used to identify the protein motif of the cation-dependent mannose 6-phosphate receptor (CD-MPR) cytoplasmic domain that is essential for these interactions. Thus, the affinity of AP-1 for membranes and in vivo transport of cathepsin D were measured for MPR-negative cells re-expressing various CD-MPR mutants. The results indicate that the targeting of lysosomal enzymes requires two distinct determinants at the carboxyl terminus of the CD-MPR cytoplasmic domain that are different from tyrosine-based endocytosis motifs. The first is a casein kinase II phosphorylation site (ESEER) that is essential for high affinity binding of AP-1 and therefore probably acts as a dominant determinant controlling CD-MPR sorting in the trans-Golgi network. The second is the adjacent di-leucine motif (HLLPM), which, by itself, is not critical for AP-1 binding, but is absolutely required for a downstream sorting event.
Footnotes
-
↵§ From the CNRS on leave from URA 1815.
-
↵* Part of this research was supported by the Association “Vaincre les Maladies Lysosomales” and the European Communities (Grant B102-CT93-02205). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵1 The abbreviations used are:
- MPR
-
mannose 6-phosphate receptor
- CD-MPR
-
cation-dependent mannose 6-phosphate receptor
- TGN
-
trans-Golgi network
- IGF
-
insulin-like growth factor
- MEM
-
minimum Eagle's medium
- DMEM
-
Dulbecco's modified Eagle's medium
- PAGE
-
polyacrylamide gel electrophoresis
- dendroaspin
-
GTP
S, guanosine 5′-O-(3-thiotriphosphate)
-
↵1H. Munier-Lehmann, F. Mauxion, U. Bauer, P. Lobel, and B. Hoflack, manuscript in preparation.
-
- Received August 25, 1995.
- Revision received November 14, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
