Bg. Yeung et Gs. Payne, Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma subunits are important for AP-1 association with clathrin coats, TRAFFIC, 2(8), 2001, pp. 565-576
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.