title: Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain.
abstract: Clathrin facilitates vesicle formation during endocytosis and sorting in the trans-Golgi network (TGN)/endosomal system. Unlike in mammals, yeast clathrin function requires both the clathrin heavy (CHC) and clathrin light (CLC) chain, since Chc1 does not form stable trimers without Clc1. To further delineate clathrin subunit functions, we constructed a chimeric CHC protein (Chc-YR) , which fused the N-terminus of yeast CHC (1-1312) to the rat CHC residues 1318-1675, including the CHC trimerization region. The novel CHC-YR allele encoded a stable protein that fractionated as a trimer. CHC-YR also complemented chc1Δ slow growth and clathrin TGN/endosomal sorting defects. In strains depleted for Clc1 (either clc1Δ or chc1Δ clc1Δ), CHC-YR, but not CHC1, suppressed TGN/endosomal sorting and growth phenotypes. Chc-YR-GFP (green fluorescent protein) localized to the TGN and cortical patches on the plasma membrane, like Chc1 and Clc1. However, Clc1-GFP was primarily cytoplasmic in chc1Δ cells harboring pCHC-YR, indicating that Chc-YR does not bind yeast CLC. Still, some partial phenotypes persisted in cells with Chc-YR, which are likely due either to loss of CLC recruitment or chimeric HC lattice instability. Ultimately, these studies have created a tool to examine non-trimerization roles for the clathrin LC.
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami, Miami, FL, USA.|Current address: Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
authors.forename: Douglas R
authors.identifier: 0000-0002-0591-443X
authors.initials: DR
authors.lastname: Boettner
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami, Miami, FL, USA.|Current Address: Department of Biology, High Point University, High Point, NC, USA.
authors.forename: Verónica A
authors.initials: VA
authors.lastname: Segarra
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami, Miami, FL, USA.
authors.forename: Balaji T
authors.initials: BT
authors.lastname: Moorthy
authors.affiliation: Departamento de Microbiologıa y Genetica/IBFG, Universidad de Salamanca/CSIC, Salamanca, Spain.
authors.forename: Nagore
authors.initials: N
authors.lastname: de León
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami, Miami, FL, USA.
authors.forename: John
authors.initials: J
authors.lastname: Creagh
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami, Miami, FL, USA.|Current address: Department of Pathology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: John R
authors.initials: JR
authors.lastname: Collette
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Miami, Miami, FL, USA.
authors.forename: Arun
authors.initials: A
authors.lastname: Malhotra
authors.affiliation: Department of Molecular and Cellular Pharmacology, University of Miami, Miami, FL, USA.
authors.forename: Sandra K
authors.initials: SK
authors.lastname: Lemmon
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D033922:Clathrin Heavy Chains / Q000235:genetics / Q000378:metabolism*; D033941:Clathrin Light Chains / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism
chemical_list: D033941:Clathrin Light Chains; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D033922:Clathrin Heavy Chains; D049452:Green Fluorescent Proteins
keywords: Clathrin; TGN/endosomal sorting; endocytosis; membrane trafficking