Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma subunits are important for AP-1 association with clathrin coats

Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo rec ruitment and clathrin assembly during clathrin-coated vesicle biogenesis. W e have identified, and characterized the physiological significance of clat hrin-binding activities in the two large subunits of the AP-1 complex in Sa ccharomyces cerevisiae. Using GST-fusion chromatography, two clathrin-bindi ng sites were defined in the beta1 subunit that match consensus clathrin-bi nding sequences in other mammalian and yeast clathrin-binding proteins. Cla thrin interactions were also identified with the C-terminal region of the g amma subunit. When introduced into chromosomal genes, point mutations in th e beta1 clathrin-binding motifs, or deletion of the gamma C-terminal region , reduced association of AP-1 with clathrin in coimmunoprecipitation assays . The beta1 mutations or the gamma truncation individually produced minor e ffects on AP-1 distribution by subcellular fractionation. However, when bet a1 and gamma mutations were combined, severe defects were observed in AP-1 association with membranes and incorporation into clathrin-coated vesicles. The combination of subunit mutations accentuated growth and alpha -factor pheromone maturation defects in chc1-ts cells, though not to the extent cau sed by complete loss of AP-1 activity. Our results suggest that both the be ta1 and gamma subunits contribute interactions with clathrin that are impor tant for stable assembly of AP-1 complexes into clathrin coats in vivo.