The ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. Much less clear is the fate of membrane-embedded molecular machines in the gas phase. Here we show that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover, subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently, we can link specific lipid and nucleotide binding with distinct regulatory roles.

Tyrosine kinase growth factor receptors and Ras/Raf/MEK/MAPK signalling have been implicated in the suppression as well as augmentation of programmed cell death. In addition, a Ras-independent role for Raf as a suppressor of programmed cell death has been suggested by the recent finding that Craf1 interacts with members of the Bcl-2 family at mitochondrial membranes. However, genetic studies of C. elegans and Drosophila, as well as the targeted mutagenesis of the murine Araf gene, have failed to support such a role. Here we show that mice with a targeted disruption in the Braf gene die of vascular defects during mid-gestation. Braf -/- embryos, unlike Araf -/- or Craf1 -/- embryos (L.W. et al., unpublished), show an increased number of endothelial precursor cells, dramatically enlarged blood vessels and apoptotic death of differentiated endothelial cells. These results establish Braf as a critical signalling factor in the formation of the vascular system and provide the first genetic evidence for an essential role of Raf gene in the regulation of programmed cell death.

No abstract

ATPases are unique rotary motors that are essential to all living organisms because of their role in energy interconversion. A three-dimensional reconstruction of the intact H+-ATPase/synthase from Thermus thermophilus has revealed the presence of two interconnected peripheral stalks, a well-defined central stalk, and a hexagonally shaped hydrophobic domain. The peripheral stalks are each attached to the water soluble sector at a noncatalytic subunit interface and extend down toward the membrane where they interact with a strong elongated tube of density that runs parallel to the membrane and connects the two stalks. The central stalk is well resolved, especially with respect to its interaction with a single catalytic subunit giving rise to an asymmetry comparable to that identified in F-ATPases. The hexagonal shape of the membrane domain might suggest the presence of 12 proteolipids arranged as dimers, analogous to the proposed arrangement in the related eukaryotic V-ATPases.