MANNOSE 6-PHOSPHATE RECEPTORS AND ADP-RIBOSYLATION FACTORS COOPERATE FOR HIGH-AFFINITY INTERACTION OF THE AP-1 GOLGI ASSEMBLY PROTEINS WITHMEMBRANES

Clathrin coat assembly in the trans-Golgi network, leading to the sequ estration of the mannose 6-phosphate receptors (MPRs) into nascent ves icles, requires the ARF-1-dependent translocation of the cytosolic AP- 1 Golgi assembly proteins onto the membranes of this organelle. The me chanistic role of the MPRs, i.e. the cargo molecules, in coat assembly is at present unclear. Using a GTP-dependent, brefeldin A-sensitive i n 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 (K-d ap proximate to 25 nM), since their number correlates the number of MPR m olecules expressed in MPR-negative cells. The quantitative electron mi croscopy shows that these high affinity binding sites are present on t rans-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 (guanos ine 5'-O-(3-triotriphosphate)), which increases the amount of membrane -bound ARF-1, mostly uncovers low affinity AP-1 binding sites (K-d app roximate to 150 nM) on trans-Golgi network membranes, normally not det ected in its absence. Collectively, these results argue that MPR sorti ng is highly coupled to the first step of coat assembly and that the M PRs, ARF-1, and possibly other proteins cooperate for high affinity in teractions of AP-1.