A genome-wide RNAi screen identifies regulators of cholesterol-modified hedgehog secretion in Drosophila

PLoS One. 2012;7(3):e33665. doi: 10.1371/journal.pone.0033665. Epub 2012 Mar 14.

Abstract

Hedgehog (Hh) proteins are secreted molecules that function as organizers in animal development. In addition to being palmitoylated, Hh is the only metazoan protein known to possess a covalently-linked cholesterol moiety. The absence of either modification severely disrupts the organization of numerous tissues during development. It is currently not known how lipid-modified Hh is secreted and released from producing cells. We have performed a genome-wide RNAi screen in Drosophila melanogaster cells to identify regulators of Hh secretion. We found that cholesterol-modified Hh secretion is strongly dependent on coat protein complex I (COPI) but not COPII vesicles, suggesting that cholesterol modification alters the movement of Hh through the early secretory pathway. We provide evidence that both proteolysis and cholesterol modification are necessary for the efficient trafficking of Hh through the ER and Golgi. Finally, we identified several putative regulators of protein secretion and demonstrate a role for some of these genes in Hh and Wingless (Wg) morphogen secretion in vivo. These data open new perspectives for studying how morphogen secretion is regulated, as well as provide insight into regulation of lipid-modified protein secretion.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • Cholesterol / pharmacology*
  • Drosophila Proteins / metabolism*
  • Drosophila melanogaster / drug effects*
  • Drosophila melanogaster / metabolism*
  • Genes, Insect / genetics
  • Genetic Testing*
  • Genome, Insect / genetics*
  • Golgi Apparatus / drug effects
  • Golgi Apparatus / metabolism
  • Golgi Apparatus / ultrastructure
  • Hedgehog Proteins / metabolism*
  • Intracellular Space / drug effects
  • Intracellular Space / metabolism
  • Luciferases, Renilla / metabolism
  • Protein Processing, Post-Translational / drug effects
  • Protein Transport / drug effects
  • RNA Interference / drug effects*
  • RNA, Double-Stranded / metabolism
  • Recombinant Fusion Proteins / metabolism
  • Reproducibility of Results
  • Subcellular Fractions / drug effects
  • Subcellular Fractions / metabolism
  • Wnt1 Protein / metabolism

Substances

  • Drosophila Proteins
  • Hedgehog Proteins
  • RNA, Double-Stranded
  • Recombinant Fusion Proteins
  • Wnt1 Protein
  • wg protein, Drosophila
  • hh protein, Drosophila
  • Cholesterol
  • Luciferases, Renilla