Systematic screens for proteins that interact with the mucolipidosis type IV protein TRPML1

PLoS One. 2013;8(2):e56780. doi: 10.1371/journal.pone.0056780. Epub 2013 Feb 13.

Abstract

Mucolipidosis type IV is a lysosomal storage disorder resulting from mutations in the MCOLN1 gene, which encodes the endosomal/lysosomal Transient Receptor Potential channel protein mucolipin-1/TRPML1. Cells isolated from Mucolipidosis type IV patients and grown in vitro and in in vivo models of this disease both show several lysosome-associated defects. However, it is still unclear how TRPML1 regulates the transport steps implicated by these defects. Identifying proteins that associate with TRPML1 will facilitate the elucidation of its cellular and biochemical functions. We report here two saturation screens for proteins that interact with TRPML1: one that is based on immunoprecipitation/mass spectrometry and the other using a genetic yeast two-hybrid approach. From these screens, we identified largely non-overlapping proteins, which represent potential TRPML1-interactors., Using additional interaction assays on some of the potential interactors from each screen, we validated some proteins as candidate TRPML1 interactors In addition, our analysis indicates that each of the two screens not only identified some false-positive interactors, as expected from any screen, but also failed to uncover potential TRPML1 interactors. Future studies on the true interactors, first identified in these screens, will help elucidate the structure and function of protein complexes containing TRPML1.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Line
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HeLa Cells
  • Humans
  • Immunoprecipitation / methods
  • Mass Spectrometry / methods
  • Mice
  • Microscopy, Confocal
  • Mucolipidoses / genetics
  • Mucolipidoses / metabolism*
  • Mutation
  • Protein Binding
  • Protein Interaction Mapping / methods*
  • Proteins / genetics
  • Proteins / metabolism*
  • Transient Receptor Potential Channels / genetics
  • Transient Receptor Potential Channels / metabolism*
  • Two-Hybrid System Techniques

Substances

  • MCOLN1 protein, human
  • Mcoln1 protein, mouse
  • Proteins
  • Transient Receptor Potential Channels
  • Green Fluorescent Proteins