The light chain subunit is required for clathrin function in Saccharomyces cerevisiae

Abstract

Clathrin, a multimeric protein involved in intracellular protein trafficking, is composed of three heavy chains (Chc) and three light chains (Clc). Upon disruption (clclΔ) of the single Clc-encoding gene (CLC1) in yeast, the steady state protein levels of Chc decreased 5-10-fold compared with wild type cells; consequently, phenotypes exhibited by clclΔ cells may result indirectly from the loss of Chc as opposed to the absence of Clc. As an approach to directly examine Clc function, clclΔ strains were generated that carry a multicopy plasmid containing the clathrin heavy chain gene (CHC1), resulting in levels of Chc 5-10-fold elevated over wild-type levels. As with deletion of CHC1, deletion of CLC1 results in defects in growth, receptor-mediated endocytosis, and maturation of the mating pheromone α- factor. However, elevated Chc expression in clclΔ cells partially suppresses the growth and α-factor maturation defects displayed by clclΔ cells alone. Biochemical analyses indicate that trimerization and assembly of Chc are perturbed in the absence of Clc, resulting in vesiculation defects. Our results demonstrate that the light chain subunit of clathrin is required for efficient Chc trimerization, proper formation of clathrin coats, and the generation of clathrin-coated vesicles.