Compromise of clathrin function and membrane association by clathrin light chain deletion.
While clathrin heavy chains from different species are highly conserved in amino acid sequence, clathrin light chains are much more divergent. Thus clathrin light chain may have different functions in different organisms. To investigate clathrin light chain function, we cloned the clathrin light chain, clcA, from Dictyostelium and examined clathrin function in clcA-mutants. Phenotypic deficiencies in development, cytokinesis, and osmoregulation showed that light chain was critical for clathrin function in Dictyostelium. In contrast with budding yeast, we found the light chain did not influence steady-state levels of clathrin, triskelion formation, or contribute to clathrin over-assembly on intracellular membranes. Imaging GFP-CHC in clcA- mutants showed that the heavy chain formed dynamic punctate structures that were remarkably similar to those found in wild-type cells. However, clathrin light chain knockouts showed a decreased association of clathrin with intracellular membranes. Unlike wild-type cells, half of the clathrin in clcA- mutants was cytosolic, suggesting that the absence of light chain compromised the assembly of triskelions onto intracellular membranes. Taken together, these results suggest a role for the Dictyostelium clathrin light chain in regulating the self-assembly of triskelions onto intracellular membranes, and demonstrate a crucial contribution of the light chain to clathrin function in vivo.
Wang
Jingshan
J
Section of Molecular Cell & Developmental Biology, Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA.
Virta
Valerie C
VC
Riddelle-Spencer
Kathryn
K
O'Halloran
Theresa J
TJ
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002455:Cell Division; D002462:Cell Membrane / Q000378:metabolism*; D002498:Centrifugation; D002850:Chromatography, Gel; D002966:Clathrin / Q000737:chemistry* / Q000378:metabolism; D033922:Clathrin Heavy Chains / Q000737:chemistry / Q000235:genetics; D033941:Clathrin Light Chains / Q000235:genetics* / Q000502:physiology; D003001:Cloning, Molecular; D003600:Cytosol / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011995:Recombination, Genetic; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D019076:Transgenes; D014867:Water / Q000737:chemistry
D002966:Clathrin; D033941:Clathrin Light Chains; D008164:Luminescent Proteins; D010455:Peptides; D014867:Water; D033922:Clathrin Heavy Chains; D049452:Green Fluorescent Proteins