Adaptor complex-independent clathrin function in yeast.
Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.
Yeung
B G
BG
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles, California 90095, USA.
Phan
H L
HL
Payne
G S
GS
D033942:Adaptor Proteins, Vesicular Transport; D002268:Carboxypeptidases / Q000378:metabolism; D043402:Cathepsin A; D002966:Clathrin / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D018699:Coated Vesicles / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics*; D011233:Precipitin Tests; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D033942:Adaptor Proteins, Vesicular Transport; D002966:Clathrin; D005656:Fungal Proteins; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D002268:Carboxypeptidases; D043402:Cathepsin A