In silico screening for palmitoyl substrates reveals a role for DHHC1/3/10 (zDHHC1/3/11)-mediated neurochondrin palmitoylation in its targeting to Rab5-positive endosomes

J Biol Chem. 2013 Jul 5;288(27):19816-29. doi: 10.1074/jbc.M112.431676. Epub 2013 May 16.


Protein palmitoylation, a common post-translational lipid modification, plays an important role in protein trafficking and functions. Recently developed palmitoyl-proteomic methods identified many novel substrates. However, the whole picture of palmitoyl substrates has not been clarified. Here, we performed global in silico screening using the CSS-Palm 2.0 program, free software for prediction of palmitoylation sites, and selected 17 candidates as novel palmitoyl substrates. Of the 17 candidates, 10 proteins, including 6 synaptic proteins (Syd-1, transmembrane AMPA receptor regulatory protein (TARP) γ-2, TARP γ-8, cornichon-2, Ca(2+)/calmodulin-dependent protein kinase IIα, and neurochondrin (Ncdn)/norbin), one focal adhesion protein (zyxin), two ion channels (TRPM8 and TRPC1), and one G-protein-coupled receptor (orexin 2 receptor), were palmitoylated. Using the DHHC palmitoylating enzyme library, we found that all tested substrates were palmitoylated by the Golgi-localized DHHC3/7 subfamily. Ncdn, a regulator for neurite outgrowth and synaptic plasticity, was robustly palmitoylated by the DHHC1/10 (zDHHC1/11; z1/11) subfamily, whose substrate has not yet been reported. As predicted by CSS-Palm 2.0, Cys-3 and Cys-4 are the palmitoylation sites for Ncdn. Ncdn was specifically localized in somato-dendritic regions, not in the axon of rat cultured neurons. Stimulated emission depletion microscopy revealed that Ncdn was localized to Rab5-positive early endosomes in a palmitoylation-dependent manner, where DHHC1/10 (z1/11) were also distributed. Knockdown of DHHC1, -3, or -10 (z11) resulted in the loss of Ncdn from Rab5-positive endosomes. Thus, through in silico screening, we demonstrate that Ncdn and the DHHC1/10 (z1/11) and DHHC3/7 subfamilies are novel palmitoyl substrate-enzyme pairs and that Ncdn palmitoylation plays an essential role in its specific endosomal targeting.

Keywords: DHHC Protein; Endosomes; Enzymes; Neurobiology; Neurochondrin; Neuroscience; Post Translational Modification; Protein Palmitoylation; Rab5.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acyltransferases / genetics
  • Acyltransferases / metabolism*
  • Animals
  • Cysteine / genetics
  • Cysteine / metabolism
  • Endosomes / genetics
  • Endosomes / metabolism*
  • Focal Adhesions / genetics
  • Focal Adhesions / metabolism
  • HEK293 Cells
  • Humans
  • Lipoylation / physiology*
  • Mice
  • Nerve Tissue Proteins / genetics
  • Nerve Tissue Proteins / metabolism*
  • Neurites / metabolism*
  • Protein Processing, Post-Translational / physiology*
  • Proteomics / methods*
  • Rats
  • Software*
  • Substrate Specificity
  • Synapses / genetics
  • Synapses / metabolism
  • rab5 GTP-Binding Proteins / genetics
  • rab5 GTP-Binding Proteins / metabolism*


  • Nerve Tissue Proteins
  • neurochondrin
  • Acyltransferases
  • ZDHHC3 protein, human
  • ZDHHC1 protein, human
  • rab5 GTP-Binding Proteins
  • Cysteine