Mannose 6-phosphate receptors and ADP-ribosylation factors cooperate for high affinity interaction of the AP-1 Golgi assembly proteins with membranes.
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 (Kd approximately 25 mM), 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 gamma S (guanosine 5'-O-(3-thiotriphosphate)), which increases the amount of membrane-bound ARF-1, most uncovers low affinity AP-1 binding sites (Kd approximately 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.
Le Borgne
R
R
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Griffiths
G
G
Hoflack
B
B
D020727:ADP-Ribosylation Factors; D033942:Adaptor Proteins, Vesicular Transport; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D002478:Cells, Cultured; D018699:Coated Vesicles / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000378:metabolism*; D012799:Sialyltransferases / Q000378:metabolism
D033942:Adaptor Proteins, Vesicular Transport; D009419:Nerve Tissue Proteins; D010455:Peptides; D010750:Phosphoproteins; D017527:Receptor, IGF Type 2; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012799:Sialyltransferases; D019204:GTP-Binding Proteins; D020727:ADP-Ribosylation Factors