No abstract

The ability to chemically couple proteins to LH(N)-fragments of clostridial neurotoxins and create novel molecules with selectivity for cells other than the natural target cell of the native neurotoxin is well established. Such molecules are able to inhibit exocytosis in the target cell and have the potential to be therapeutically beneficial where secretion from a particular cell plays a causative role in a disease or medical condition. To date, these molecules have been produced by chemical coupling of the LH(N)-fragment and the targeting ligand. This is, however, not a suitable basis for producing pharmaceutical agents as the products are ill defined, difficult to control and heterogeneous. Also, the molecules described to date have targeted neuroendocrine cells that are susceptible to native neurotoxins, and therefore the benefit of creating a molecule with a novel targeting domain has been limited. In this paper, the production of a fully recombinant fusion protein from a recombinant gene encoding both the LH(N)-domain of a clostridial neurotoxin and a specific targeting domain is described, together with the ability of such recombinant fusion proteins to inhibit secretion from non-neuronal target cells. Specifically, a novel protein consisting of the LH(N)-domains of botulinum neurotoxin type C and epidermal growth factor (EGF) that is able to inhibit secretion of mucus from epithelial cells is reported. Such a molecule has the potential to prevent mucus hypersecretion in asthma and chronic obstructive pulmonary disease.

Vacuolar H(+)-ATPases (V-ATPases) are multi-subunit membrane proteins that couple ATP hydrolysis to the extrusion of protons from the cytoplasm. Although they share a common macromolecular architecture and rotational mechanism with the F(1)F(0)-ATPases, the organization of many of the specialized V-ATPase subunits within this rotary molecular motor remains uncertain. In this study, we have identified sequence segments involved in linking putative stator subunits in the Saccharomyces V-ATPase. Precipitation assays revealed that subunits Vma5p (subunit C) and Vma10p (subunit G), expressed as glutathione-S-transferase fusion proteins in E. coli, are both able to interact strongly with Vma4p (subunit E) expressed in a cell-free system. GST-Vma10p also associated with Vma2p and Vma1p, the core subunits of the ATP-hydrolyzing domain, and was able to self-associate to form a dimer. Mutations within the first 19-residue region of Vma4p, which disrupted interaction with Vma5p in vitro, also prevented the Vma4p polypeptide from restoring V-ATPase function in a complementation assay in vivo. These mutations did not prevent assembly of Vma5p (subunit C) and Vma2p (subunit B) into an inactive complex at the vacuolar membrane, indicating that Vma5p must make multiple interactions involving other V-ATPase subunits. A second, highly conserved region of Vma4p between residues 19 and 38 is involved in binding Vma10p. This region is highly enriched in charged residues, suggesting a role for electrostatic effects in Vma4p-Vma10p interaction. These protein interaction studies show that the N-terminal region of Vma4p is a key factor not only in the stator structure of the V-ATPase rotary molecular motor, but also in mediating interactions with putative regulatory subunits.

To operate as a rotary motor, the ATP-hydrolyzing domain of the vacuolar H(+)-ATPase must be connected to a fixed structure in its membrane-bound proton pump domain by a mechanical stator. Although low-resolution structural data and spectroscopic analysis indicate that a filament-like subunit E/subunit G heterodimer performs this role, more detailed information about the relative arrangement of these subunits is limited. We have used a site-directed cross-linking approach to show that, in both bacterial and yeast V-type ATPases, the N-terminal alpha-helical segments of the G and E subunits are closely aligned over a distance of up to 40 A. Furthermore, cross-linking coupled to mass spectrometry shows that the C-terminal end of G is anchored at the C-terminal globular domain of subunit E. These data are consistent with a stator model comprising two approximately 150 A long parallel alpha-helices linked to each other at both ends, stabilized by a coiled-coil arrangement and capped by the globular C-terminal domain of E that connects the cytoplasmic end of the helical structure to the V-ATPase catalytic domain.

In the absence of a high-resolution structure for the vacuolar H+-ATPase, a number of approaches can yield valuable information about structure/function relationships in the enzyme. Electron microscopy can provide not only a representation of the overall architecture of the complex, but also a low-resolution map onto which structures solved for individually expressed subunits can be fitted. Here we review the possibilities for electron microscopy of the Saccharomyces V-ATPase and examine the suitability of V-ATPase subunits for expression in high yield prokaryotic systems, a key step towards high-resolution structural studies. We also review the role of experimentally-derived structural models in understanding structure/function relationships in the V-ATPase, with particular reference to the complex of proton-translocating 16 kDa proteolipids in the membrane domain of the V-ATPase. This model in turn makes testable predictions about the sites of binding of bafilomycins and the functional interactions between the proteolipid and the single-copy membrane subunit Vph1p, with implications for the constitution of the proton translocation pathway.