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 (clc1⌬) 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 clc1⌬ cells may result indirectly from the loss of Chc as opposed to the absence of Clc. As an approach to directly examine Clc function, clc1⌬ 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 ␣-factor. However, elevated Chc expression in clc1⌬ cells partially suppresses the growth and ␣-factor maturation defects displayed by clc1⌬ 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.Distinct compartments in eukaryotic cells are maintained through the selective transport of proteins carried out by small vesicular carriers. Generation of these vesicles involves assembly of proteinaceous coats on the cytoplasmic surface of donor compartment membranes, which leads to the budding of coated vesicles. Although a variety of proteins are transported through vesicular movement, a subset of specific trafficking events are mediated by clathrin-coated vesicles. These include the retention of resident Golgi membrane proteins, receptormediated endocytosis, and the sorting of lysosomal/vacuolar proteins from the secretory pathway to the lysosome/vacuole (1, 2).The clathrin molecule, or triskelion, is a hexamer composed of three heavy chain subunits (Chc) and three light chain subunits (Clc) (1). In mammals, there are two forms of light chain, LC a and LC b , which share 60% amino acid identity and appear to be randomly distributed in clathrin trimers. In yeast, there is only one form of light chain encoded by the CLC1 gene (3). Formation of a clathrin-coated vesicle is initiated by binding of clathrin-associated protein complexes (APs) 1 to the donor membrane. Triskelions associate with the APs and polymerize into polygonal lattice structures. Such clathrin-coated membrane segments, known as coated pits, are thought to collect specific cargo proteins through interactions between the cargo protein cytoplasmic domains and the AP complexes. The clathrin-coated pit then invaginates and pinches off to form a clathrin-coated vesicle carrying the selected cargo proteins. Formation of the vesicle may involve rearrangement of subunits assembled on the coated pit, or it may be driven by the polymerization of new clathrin subunits into a polyhe...

Novel clathrin activity: developments in health and disease

“…The structure showed that the molecules in the asymmetric unit were trimeric, but threefold symmetry was disturbed (68). This observation supports previous findings that CLC contributes to the net stability of trimeric clathrin (69)(70)(71).…”

“…Additional work will be needed to sort out whether the position of helix 7j depends at all on the C-terminus of CLC. The fact that CLC knockouts in Saccharomyces cerevisiae destroy the trimeric clathrin structure (70,71), while the clathrin structure in Dicytostelium is unaffected by knocking down CLC (73), suggests that any CLC role will likely be organism specific.…”

Endocytic proteins drive vesicle growth via instability in high membrane tension environment

“…In yeast cells, clathrin, actin, and BAR proteins contribute to vesicle formation in different capacities. Though the inhibition of actin polymerization completely arrests endocytosis (9,11,12,14), the absence of clathrin and BAR proteins only leads to ∼50% and 25% reduction in the internalization events, respectively (14)(15)(16)(17)(18). Although a high scission rate is maintained in BAR mutant cells, there is a fundamental difference between the shape evolution process in these and the wild-type cells.…”

Direct interaction of Cbl with pTyr 1045 of the EGF receptor (EGFR) is required to sort the EGFR to lysosomes for degradation

“…The CLC exists in two isoforms, LCa and LCb. These appear to be randomly recruited to clathrin triskelia and are believed to be involved in CHC trimerization (Chu et al, 1996;Huang et al, 1997).…”