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Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain
Author(s) -
Boettner Douglas R.,
Segarra Verónica A.,
Moorthy Balaji T.,
León Nagore,
Creagh John,
Collette John R.,
Malhotra Arun,
Lemmon Sandra K.
Publication year - 2016
Publication title -
traffic
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.677
H-Index - 130
eISSN - 1600-0854
pISSN - 1398-9219
DOI - 10.1111/tra.12401
Subject(s) - clathrin , biology , immunoglobulin light chain , yeast , microbiology and biotechnology , heavy chain , chain (unit) , clathrin adaptor proteins , computational biology , endocytosis , genetics , gene , receptor , physics , astronomy , antibody
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 p CHC‐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.