Genome-wide siRNA screen identifies UNC50 as a regulator of Shiga toxin 2 trafficking

J Cell Biol. 2017 Oct 2;216(10):3249-3262. doi: 10.1083/jcb.201704015. Epub 2017 Sep 7.

Abstract

Shiga toxins 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol. Early endosome-to-Golgi transport allows the toxins to evade degradation in lysosomes. Targeting this trafficking step has therapeutic promise, but the mechanism of trafficking for the more potent toxin STx2 is unclear. To identify host factors required for early endosome-to-Golgi trafficking of STx2, we performed a viability-based genome-wide siRNA screen in HeLa cells. 564, 535, and 196 genes were found to be required for toxicity induced by STx1 only, STx2 only, and both toxins, respectively. We focused on validating endosome/Golgi-localized hits specific for STx2 and found that depletion of UNC50 blocked early endosome-to-Golgi trafficking and induced lysosomal degradation of STx2. UNC50 acted by recruiting GBF1, an ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF), to the Golgi. These results provide new information about STx2 trafficking mechanisms and may advance efforts to generate therapeutically viable toxin-trafficking inhibitors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Endosomes / genetics
  • Endosomes / metabolism
  • Genome-Wide Association Study*
  • Golgi Apparatus / genetics
  • Golgi Apparatus / metabolism
  • HeLa Cells
  • Humans
  • Lysosomes / genetics
  • Lysosomes / metabolism
  • Membrane Proteins* / genetics
  • Protein Transport / genetics
  • Proteolysis
  • RNA, Small Interfering*
  • RNA-Binding Proteins* / genetics
  • Shiga Toxin 1 / genetics
  • Shiga Toxin 1 / metabolism
  • Shiga Toxin 2 / genetics
  • Shiga Toxin 2 / metabolism*

Substances

  • Membrane Proteins
  • RNA, Small Interfering
  • RNA-Binding Proteins
  • Shiga Toxin 1
  • Shiga Toxin 2
  • UNC50 protein, human