Clathrin Interactions with C‐Terminal Regions of the Yeast AP‐1 β and γ Subunits are Important for AP‐1 Association with Clathrin Coats

Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo recruitment and clathrin assembly during clathrin‐coated vesicle biogenesis. We have identified, and characterized the physiological significance of clathrin‐binding activities in the two large subunits of the AP‐1 complex in Saccharomyces cerevisiae . Using GST‐fusion chromatography, two clathrin‐binding sites were defined in the β1 subunit that match consensus clathrin‐binding sequences in other mammalian and yeast clathrin‐binding proteins. Clathrin interactions were also identified with the C‐terminal region of the γ subunit. When introduced into chromosomal genes, point mutations in the β1 clathrin‐binding motifs, or deletion of the γ C‐terminal region, reduced association of AP‐1 with clathrin in coimmunoprecipitation assays. The β1 mutations or the γ truncation individually produced minor effects on AP‐1 distribution by subcellular fractionation. However, when β1 and γ 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 α‐factor pheromone maturation defects in chc1‐ts cells, though not to the extent caused by complete loss of AP‐1 activity. Our results suggest that both the β1 and γ subunits contribute interactions with clathrin that are important for stable assembly of AP‐1 complexes into clathrin coats in vivo .