In vivo dynamics of clathrin and its adaptor-dependent recruitment to the actin-based endocytic machinery in yeast.
Clathrin-mediated transport is a major pathway for endocytosis. However, in yeast, where cortical actin patches are essential for endocytosis, plasma membrane-associated clathrin has never been observed. Using live cell imaging, we demonstrate cortical clathrin in association with the actin-based endocytic machinery in yeast. Fluorescently tagged clathrin is found in highly mobile internal trans-Golgi/endosomal structures and in smaller cortical patches. Total internal reflection fluorescence microscopy showed that cortical patches are likely endocytic sites, as clathrin is recruited prior to a burst of intensity of the actin patch/endocytic marker, Abp1. Clathrin also accumulates at the cortex with internalizing alpha factor receptor, Ste2p. Cortical clathrin localizes with epsins Ent1/2p and AP180s, and its recruitment to the surface is dependent upon these adaptors. In contrast, Sla2p, End3p, Pan1p, and a dynamic actin cytoskeleton are not required for clathrin assembly or exchange but are required for the mobility, maturation, and/or turnover of clathrin-containing endocytic structures.
Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Thomas M
TM
Newpher
Robin P
RP
Smith
Vance
V
Lemmon
Sandra K
SK
Lemmon
D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D033942:Adaptor Proteins, Vesicular Transport; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002966:Clathrin / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000235:genetics / Q000378:metabolism; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D034041:Monomeric Clathrin Assembly Proteins / Q000378:metabolism; D009154:Mutation; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D014157:Transcription Factors / Q000378:metabolism; D033921:Vesicular Transport Proteins
C062612:ABP1 protein, S cerevisiae; D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002352:Carrier Proteins; D002966:Clathrin; D003598:Cytoskeletal Proteins; C121319:ENT1 protein, S cerevisiae; C121321:ENT2 protein, S cerevisiae; D008840:Microfilament Proteins; D034041:Monomeric Clathrin Assembly Proteins; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; C082266:SLA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013844:Thiazoles; D053778:Thiazolidines; D014157:Transcription Factors; D033921:Vesicular Transport Proteins; C439023:clathrin assembly protein AP180; C037067:latrunculin A