Mannose 6-Phosphate Receptors and ADP-ribosylation Factors Cooperate for High Affinity Interaction of the AP-1 Golgi Assembly Proteins with Membranes (*)
- From the European Molecular Biology Laboratory, Postfach 10-2209, Meyerhofstrasse 1, 69012 Heidelberg, Federal Republic of Germany
- § To whom correspondence should be addressed. Tel.: 49-6221-387-285; Fax: 49-6221-387-306.
Abstract
Clathrin coat assembly in the trans-Golgi network, leading to the sequestration of the mannose 6-phosphate receptors (MPRs)
into nascent vesicles, requires the ARF-1-dependent translocation of the cytosolic AP-1 Golgi assembly proteins onto the membranes
of this organelle. The mechanistic role of the MPRs, i.e. the cargo molecules, in coat assembly is at present unclear. Using a GTP-dependent, brefeldin A-sensitive in vitro AP-1 binding assay, we have determined here the parameters of the AP-1 binding reaction. We demonstrate that, in addition
of ARF-1, the MPRs contribute to create high affinity AP-1 binding sites (K
≈ 25 nM), since their number correlates the number of MPR molecules expressed in MPR-negative cells. The quantitative electron
microscopy shows that these high affinity binding sites are present on trans-Golgi network membranes, as expected, and to
some extent on early endosomes. The high affinity binding sites are lost when the MPRs or ARF-1 become rate-limiting components.
Conversely, GTP
S (guanosine 5′-O-(3-thiotriphosphate)), which increases the amount of membrane-bound ARF-1, mostly uncovers low affinity AP-1 binding sites
(K
≈ 150 nM) on trans-Golgi network membranes, normally not detected in its absence. Collectively, these results argue that
MPR sorting is highly coupled to the first step of coat assembly and that the MPRs, ARF-1, and possibly other proteins cooperate
for high affinity interactions of AP-1.
Footnotes
-
↵*This work was supported in part by grants from “Vaincre les Maladies Lysosomales” and NATO(900226) and by the European communities (Grant B102-CT93-0205). 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:
- TGN
-
trans-Golgi network
- IGF
-
insulin-like growth factor
- MPR
-
mannose 6-phosphate receptor
- GTP
S -
guanosine 5′-O-(3-thiotriphosphate)
- NRK
-
normal rat kidney
- PBS
-
phosphate-buffered saline
- ELISA
-
enzyme-linked immunosorbent assay
- BFA
-
brefeldin A
- VSV-G
-
vesicular stomatitis virus-G protein
- BSA
-
bovine serum albumin
- SLO
-
streptolysin O.
-
- Received August 25, 1995.
- Revision received November 14, 1995.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
