MANNOSE 6-PHOSPHATE RECEPTORS REGULATE THE FORMATION OF CLATHRIN-COATED VESICLES IN THE TGN

The transport of the two mannose 6-phosphate receptors (MPRs) from the secretory pathway to the endocytic pathway is mediated by carrier ves icles coated with the AP-1 Golgi-specific assembly protein and clathri n. Using an in vitro assay that reconstitutes the ARF-l-dependent tran slocation of cytosolic AP-1 onto membranes of the TGN, we have previou sly reported that the MPRs are key components for the efficient recrui tment of AP-1 (Le Borgne, R., G. Griffiths, and B. Hoflack. 1996. J. B iol. Chem. 271:2162-2170). Using a polyclonal antibody against the mou se gamma-adaptin, we have now examined the steady state distribution o f AP-1 after subcellular fractionation of mouse fibroblasts lacking bo th MPRs or reexpressing physiological levels of either MPR, We report that the amount of AP-1 bound to membranes and associated with clathri n-coated vesicles depends on the expression level of the MPRs and on t he integrity of their cytoplasmic domains. Thus, these results indicat e that the concentration of the MPRs, i.e., the major transmembrane pr oteins sorted toward the endosomes, determines the number of clathrin- coated Vesicles formed in the TGN.