Sorting of Lysosomal Membrane Glycoproteins lamp-1 and lamp-2 into Vesicles Distinct from Mannose 6-Phosphate Receptor/γ-Adaptin Vesicles at the trans-Golgi Network*
- From the Department of Medical Biochemistry and Biophysics, University of Umeå, S-901 87 Umeå, Sweden
Abstract
Newly synthesized lysosomal membrane glycoproteins lamp-1 and lamp-2 are primarily sorted at thetrans-Golgi network (TGN) by recognition of a tyrosine-based signal sequence in their cytoplasmic tails. It is presently unclear how this signal is recognized and what type of vesicle transports lamp-1 and lamp-2. Here, we describe a method to generate transport vesicles containing lamp proteins from the TGNin vitro. The method is based on incorporation of radioactive sialic acid in glycoproteins at the TGN by incubation of membranes with tritiated CMP-sialic acid. The generation of vesicles from labeled membranes required ATP and cytosol, and was temperature-dependent and brefeldin A-sensitive. Analysis on Nycodenz gradients revealed that lamp-vesicles were distinct from vesicles containing γ-adaptin and mannose 6-phosphate receptor (MPR). Moreover, both these types of vesicles migrated differently than vesicles containing proteins destined for the plasma membrane. The conclusion that lamps and MPRs are sorted into different vesicles was further strengthened by the finding that whereas wortmannin bothin vitro and in vivo inhibited the production of γ-adaptin/MPR-containing vesicles, this drug had no effect on the generation of lamp-vesicles and on the sorting of lamps. The results indicate that membrane proteins containing tyrosine-based motifs for sorting at the TGN are segregated from clathrin-coated vesicles containing MPRs.
Footnotes
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↵* This work was supported by Swedish Medical Research Council Grant 03X-07886 and by the Medical Faculty at Umeå University.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 all correspondence should be addressed: Dept. of Medical Biochemistry and Biophysics, University of Umeå, S-901 87 Umeå, Sweden. Tel.: 46-90-7866743; Fax: 46-90-136310; E-mail:sven.carlsson{at}medkem.umu.se.
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↵1 The abbreviations used are: TGN,trans-Golgi network; AP, adaptor protein; ARF, ADP-ribosylation factor; CCV, clathrin-coated vesicle; GTPγS, guanosine 5′-3-O-(thio)triphosphate; FITC, fluorescein isothiocyanate; lamp, lysosome-associated membrane protein; MPR, mannose 6-phosphate receptor; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; PNS, post-nuclear supernatant.
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- Received March 17, 1998.
- Revision received May 8, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
