Fluorescence mapping of mitochondrial TIM23 complex reveals a water-facing, substrate-interacting helix surface

Cell. 2008 Aug 8;134(3):439-50. doi: 10.1016/j.cell.2008.06.007.

Abstract

Protein translocation across the mitochondrial inner membrane is mediated by the TIM23 complex. While its central component, Tim23, is believed to form a protein-conducting channel, the regions of this subunit that face the imported protein are unknown. To examine Tim23 structure and environment in intact membranes at high resolution, various derivatives, each with a single, environment-sensitive fluorescent probe positioned at a specific site, were assembled into functional TIM23 complexes in active mitochondria and analyzed by multiple spectral techniques. Probes placed sequentially throughout a transmembrane region that was identified by crosslinking as part of the protein-conducting channel revealed an alpha helix in an amphipathic environment. Probes on the aqueous-facing helical surface specifically underwent spectral changes during protein import, and their accessibility to hydrophilic quenching agents is considered in terms of channel gating. This approach has therefore provided an unprecedented view of a translocon channel structure in an intact, fully operational, membrane-embedded complex.

Publication types

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

MeSH terms

  • Membrane Transport Proteins / chemistry*
  • Membrane Transport Proteins / metabolism
  • Mitochondria / chemistry
  • Mitochondria / metabolism
  • Mitochondrial Membranes / chemistry*
  • Mitochondrial Membranes / metabolism*
  • Mitochondrial Proteins / metabolism
  • Multiprotein Complexes / chemistry
  • Protein Structure, Secondary
  • Protein Transport
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Spectrometry, Fluorescence / methods*

Substances

  • Membrane Transport Proteins
  • Mitochondrial Proteins
  • Multiprotein Complexes
  • Saccharomyces cerevisiae Proteins
  • TIM23 protein, S cerevisiae