Forum 03/06/2003

“Approaches to Time-Resolved Electron Cryo-Microscopy”

Howard White – John Rubinstein Lab, Dept of Biochemistry, Univ of Toronto

Vacoular-type ATPases (V-ATPases) in eukaryotes are ATP-dependent proton pumps that acidify intracellular compartments such as lysosomes, endosomes, and secretory vesicles.  V-ATPase is evolutionarily related to the F-type ATP synthase but they differ in subunit composition and arrangement.  Both enzymes use a rotary catalytic mechanism where ATP hydrolysis drives rotation of a rotor subcomplex within the enzyme resulting in proton translocation through the membrane-bound Fo or Vo regions.  The reaction can also run in the opposite direction during ATP synthesis.  In some eubacteria and archaea, V-ATPase functions exclusively as an ATP synthase in order to generate ATP for these organisms.  The overall architecture of V-ATPase can be separately into a soluble, catalytic V1 region, a membrane-bound region as well as the peripheral stalks.  Here we report the intact structure of the V-ATPase from the eubacterium Thermus thermophilus determined by single particle electron cryomicroscopy (cryo-EM).  The map can be segmented into individual subunits and this allows rigid-body and flexible fitting of atomic models into their corresponding subunit map densities.  Asymmetric features in the resulting structural model suggest that the T. thermophilus V-ATPase relaxes to a single overall conformation when not performing ATP synthesis or hydrolysis.  Most significantly, the interactions of subunits in the membrane-bound region provide insights into the mechanism of proton translocation, which is currently unknown due to the absence of high resolution X-ray crystal structures available for this region.

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