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 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 beta1 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 gamma subunit. When introduced into chromosomal genes, point mutations in the 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 effects on AP-1 distribution by subcellular fractionation. However, when beta1 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 caused by complete loss of AP-1 activity. Our results suggest that both the beta1 and gamma subunits contribute interactions with clathrin that are important for stable assembly of AP-1 complexes into clathrin coats in vivo.
Yeung
B G
BG
Department of Biological Chemistry, UCLA School of Medicine, Los Angeles, CA 90095-1737, USA.
Payne
G S
GS
D033966:Adaptor Protein Complex beta Subunits; D000483:Alleles; D020816:Amino Acid Motifs; D001665:Binding Sites; D002455:Cell Division; D002966:Clathrin / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D000072235:Mating Factor; D010455:Peptides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*
C429285:APL2 protein, S cerevisiae; D033966:Adaptor Protein Complex beta Subunits; D002966:Clathrin; D010455:Peptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D018808:Transcription Factor AP-1; D000072235:Mating Factor; D005982:Glutathione Transferase