Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain

Douglas R. Boettner, Verónica A. Segarra, Balaji T. Moorthy, Nagore de León, John Creagh, John R. Collette, Arun Malhotra, Sandra Lemmon

Research output: Contribution to journalArticle

2 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)754-768
Number of pages15
JournalTraffic
Volume17
Issue number7
DOIs
StatePublished - Jul 1 2016

Fingerprint

Clathrin Light Chains
Clathrin Heavy Chains
Clathrin
Yeast
trans-Golgi Network
Yeasts
Sorting
Phenotype
Growth
Mammals
Endocytosis
Green Fluorescent Proteins
Cell membranes
Proteins
Alleles
Rats
Cell Membrane
Defects

Keywords

  • Clathrin
  • endocytosis
  • membrane trafficking
  • TGN/endosomal sorting

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Structural Biology
  • Molecular Biology
  • Genetics

Cite this

Boettner, D. R., Segarra, V. A., Moorthy, B. T., de León, N., Creagh, J., Collette, J. R., ... Lemmon, S. (2016). Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain. Traffic, 17(7), 754-768. https://doi.org/10.1111/tra.12401

Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain. / Boettner, Douglas R.; Segarra, Verónica A.; Moorthy, Balaji T.; de León, Nagore; Creagh, John; Collette, John R.; Malhotra, Arun; Lemmon, Sandra.

In: Traffic, Vol. 17, No. 7, 01.07.2016, p. 754-768.

Research output: Contribution to journalArticle

Boettner, DR, Segarra, VA, Moorthy, BT, de León, N, Creagh, J, Collette, JR, Malhotra, A & Lemmon, S 2016, 'Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain', Traffic, vol. 17, no. 7, pp. 754-768. https://doi.org/10.1111/tra.12401
Boettner DR, Segarra VA, Moorthy BT, de León N, Creagh J, Collette JR et al. Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain. Traffic. 2016 Jul 1;17(7):754-768. https://doi.org/10.1111/tra.12401
Boettner, Douglas R. ; Segarra, Verónica A. ; Moorthy, Balaji T. ; de León, Nagore ; Creagh, John ; Collette, John R. ; Malhotra, Arun ; Lemmon, Sandra. / Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain. In: Traffic. 2016 ; Vol. 17, No. 7. pp. 754-768.
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