INHIBITION OF CALCIUM-INDEPENDENT MANNOSE 6-PHOSPHATE RECEPTOR INCORPORATION INTO TRANS-GOLGI NETWORK-DERIVED CLATHRIN-COATED VESICLES BY WORTMANNIN

The transport of pro-catepsin D from the trans-Golgi network (TGN) to the endosomal pathway is dependent on binding to the calcium-independe nt mannose 6-phosphate receptor (ci-M6PR), which is incorporated into TGN-derived clathrin-coated transport vesicles (CCVs). Inhibition of t his transport step by wortmannin has led to the proposal that it is de pendent upon a phosphoinositide 3-kinase activity necessary for ci-M6P R recruitment into TGM-derived CCVs or in the formation of those vesic les (Brown, W., J,, DeWald, D, B,, Emr, S. D,, Plutner, H., and Balch, W, E, (1995) J. Cell Biol. 130, 781-796; Davidson, H, W. (1995) J. Ce ll Biol. 130, 781-796), In this study we have addressed the effect of wortmannin on the TGN step of the ci-M6PR, cycle, CCVs from H562 cells , pretreated or not with 250 nM wortmannin, were purified on equilibri um density gradients. Quantification of TGN-derived CCVs, assessed by gamma-adaptin content in purified vesicle fractions, showed that the f ormation of the vesicles was only marginally decreased after 20 min of treatment with the drug, while for the same wortmannin treatment, the drug of ci-M6PR recruited into those vesicles was decreased by 70% co mpared with control, At a later time point (2 h), a reduction in the a mount of gamma-adaptin in CCV fractions was also observed, These findi ngs demonstrate that inhibition of ci-M6PR recruitment into CCVs but n ot of vesicle formation is the primary reason for the observed defect in cathepsin D transport following wortmannin treatment.