Characterization of a fourth adaptor-related protein complex

Adaptor protein complexes (APs) function as vesicle coat components in diff erent membrane traffic pathways; however, there are a number of pathways fo r which there is still no candidate coat. To find novel coat components rel ated to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitati on and yeast two-hybrid analysis that these four proteins (epsilon, beta 4, mu 4, and sigma 4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 i s localized to similar to 10-20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with me mbranes is regulated by the small GTPase ARF. Immunogold electron microscop y indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The mu 4 subunit of the complex specifi cally interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sort ing of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participat es in a specialized trafficking pathway but one that is required in all cel l types.