AP-1 and AP-3 mediate sorting of melanosomal and lysosomal membrane proteins into distinct post-Golgi trafficking pathways.
The adaptor complexes AP-1 and AP-3 are localized to endosomes and/or the trans Golgi network (TGN). Because of limitations in analysing intracellular adaptor function directly, their site of function is a matter of ongoing uncertainty. To overcome this problem and to analyse adaptor sorting at the TGN, we reconstituted vesicle formation from Golgi/TGN-enriched membranes in a novel in vitro budding assay. Melanocytes were metabolically labelled followed by a 19 degrees C temperature block to accumulate newly synthesized proteins in Golgi membranes, which were then enriched by subcellular fractionation and used as donor membranes for vesicle formation in vitro. The incorporation of the melanosomal proteins tyrosinase and tyrosinase-related protein 1 (TRP-1) as well as Lamp-1 and 46 kDa mannose-6-phosphate receptor (MPR46) into Golgi/TGN-derived vesicles was temperature, nucleotide, cytosol, ADP ribosylation factor 1 and adaptor dependent. We show that sorting of TRP-1 and MPR46 was AP-1 dependent, while budding of tyrosinase and Lamp-1 required AP-3. Depletion of clathrin inhibited sorting of all four cargo proteins, suggesting that AP-1 and AP-3 are involved in the formation of distinct types of clathrin-coated vesicles, each of which is characterized by the incorporation of specific cargo membrane proteins.
Chapuy
Björn
B
Institute of Biochemistry II, Department of Hematology and Oncology, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany.
Tikkanen
Ritva
R
Mühlhausen
Chris
C
Wenzel
Dirk
D
von Figura
Kurt
K
Höning
Stefan
S
D033961:Adaptor Protein Complex 1 / Q000378:metabolism*; D033963:Adaptor Protein Complex 3 / Q000378:metabolism*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D013347:Subcellular Fractions / Q000378:metabolism; D013552:Swine
D033961:Adaptor Protein Complex 1; D033963:Adaptor Protein Complex 3; D002966:Clathrin; D052118:Lysosomal-Associated Membrane Protein 1