Binding of AP-1 Golgi adaptors to membranes requires phosphorylated cytoplasmic domains of the mannose 6-phosphate/insulin-like growth factor II receptor.
In mammalian cells, clathrin-coated vesicles mediate transport of the lysosomal enzyme receptors from the trans-Golgi network to the endocytic pathway. A critical step of this process is the recruitment of Golgi-specific adaptors onto Golgi membranes for efficient clathrin polymerization. An in vitro assay was used here to quantitate this event in streptolysin-O-permeabilized NRK cells. At 37 degrees C, these interactions are cytosol- and energy-dependent, sensitive to GTP gamma S (guanosine 5'-O-(thiotriphosphate)) and brefeldin A. We report that Golgi-specific adaptor binding is enhanced in mannose 6-phosphate/insulin-like growth factor II (IGF II) receptor-overexpressing cells and reduced in mannose 6-phosphate receptor-deficient cells. Furthermore, adaptor binding is partially inhibited after addition of soluble cytoplasmic domains of the mannose 6-phosphate/IGF II receptor. Almost complete inhibition is only observed when this domain is phosphorylated on serines 2421 and 2492, a major modification acquired during exit of the receptor from the Golgi. These results show that the mannose 6-phosphate/IGF II receptor is part of the components that recruit the Golgi-specific adaptors and that its phosphorylation is an important feature for high affinity interactions with sorting components.
Le Borgne
R
R
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Schmidt
A
A
Mauxion
F
F
Griffiths
G
G
Hoflack
B
B
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D010766:Phosphorylation; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000378:metabolism*
D002966:Clathrin; D016755:Proto-Oncogene Proteins c-jun; D017527:Receptor, IGF Type 2; D016244:Guanosine 5'-O-(3-Thiotriphosphate)