About the Project
The V-ATPase is a large and enormously complex membrane protein complex found in virtually all eukaryotic cells. It acidifies intracellular compartments and is involved in maintaining cytoplasmic pH, so is crucial for cellular function. Although fundamentally similar to the mitochondrial ATP synthase, the V-ATPase is a fantastically complicated and sophisticated molecular motor – in effect an ATP-driven proton-pumping turbine linked to an elaborate system of control rods and levers (see Muench et al 2009, details below). Interacts with the cytoskeleton control its location, and interfaces with protein kinase-mediated signalling systems in the cytoplasm regulate its activity.
In higher eukaryotes such as humans, the situation is even more complicated – most of the V-ATPase’s 13 subunits exist as multiple isoforms. Although the V-ATPase is found in most cells only in intracellular membranes, two specific isoforms are found at the surface of cancer cells. This plasma membrane location correlates with tumour cell invasiveness, and may be linked to survival of cancer cells during the early phases of metastasis, the process by which they break off from a primary tumour and invade a secondary site. Complications from metastases are a major cause of mortality.
This project has four key aims:
1. To confirm the link between plasma membrane expression of specific V-ATPase isoforms and invasiveness both in vitro and in vivo.
2. To investigate the cellular mechanism of V-ATPase/cytoskeleton interaction and targetting to the plasma membrane.
3. To determine if V-ATPase inhibitor compounds exert differential effects on different V-ATPase isoforms.
4. To investigate the mechanism of control of V-ATPase activity in tumour cells: do stimulatory signals lead to increased invasiveness?
The project has the potential to identify new biomarkers of metastatic potential and new targets for cancer therapy, and will use state-of-the-art bioimaging (live cell imaging in both cultured cells and tissues, 3D reconstructions etc) and molecular genetic methods (gene knock-outs and knock-ins, shRNA, gene expression profiling).
References
JONES RPO, DUROSE LJ, PHILLIPS C, KEEN JN, FINDLAY JBC & HARRISON MA (2010) A site-directed cross-linking approach to the characterization of subunit E-subunit G contacts in the vacuolar H+-ATPase stator. Mol. Memb. Biol. 27, 147–159
MUENCH SP, HUSS M, SONG CF, PHILLIPS C, WIECZOREK H, TRINICK J & HARRISON MA (2009) Cryo-electron microscopy of the vacuolar ATPase motor reveals its mechanical and regulatory complexity. J. Mol. Biol. 386, 989-999. doi:10.1016/j.jmb.2009.01.014
KÓTA Z, PÁLI, T, DIXON N, KEE T, HARRISON M, FINDLAY JBC, FINBOW ME & MARSH D (2008) Incorporation of transmembrane peptides from the vacuolar H+-ATPase in phospholipid membranes: spin-label electron paramagnetic resonance and polarised infrared spectroscopy. Biochemistry 47, 3937-3949
DIXON N, PALI T, KEE TP, BALL SK, HARRISON MA, FINDLAY JBC, NYMAN J, VÄÄNÄNEN K, FINBOW ME & MARSH D (2008) Interaction of spin-labelled inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector. Biophys. J. 94, 506–514. doi: 10.1529/biophysj.107.111781
DUARTE AMS, WOLFS CJAM, VAN NULAND NAJ, HARRISON MA, FINDLAY JBC, VAN MIERLO CPM & HEMMINGA MA (2007) Structure and localization of an essential transmembrane segment of the proton translocation channel of yeast H+-V-ATPase. Biophys. Biochim. Acta (Biomembranes) 1768, 218-227.
CLARE DK, ORLOVA EV, FINBOW ME, HARRISON MA, FINDLAY JBC, & SAIBIL HR (2006) An expanded and flexible form of the vacuolar ATPase membrane sector. Structure 14, 1149-1156. DOI:10.1016/j.str.2006.05.014
WHYTESIDE G, MEEK PJ, BALL SK, DIXON N, FINBOW ME, KEE TP, FINDLAY JBC & HARRISON MA (2005) Concanamycin and indolyl pentadieneamide inhibitors of the vacuolar H+-ATPase bind with high affinity to the purified proteolipid subunit of the membrane domain. Biochemistry 44, 15024 -15031. DOI: 10.1021/bi051529h
JONES RPO, DUROSE, L, FINDLAY JBC & HARRISON MA (2005) Defined sites of interaction between subunits E (Vma4p), C (Vma5p) and G (Vma10p) within the stator structure of the vacuolar H+-ATPase. Biochemistry 44, 3933-3941. DOI: 10.1021/bi048402x
PALI T, WHYTESIDE G, DIXON N, KEE TP, BALL S, HARRISON MA, FINDLAY JBC, FINBOW ME & MARSH D (2004) Interaction of inhibitors of the vacuolar H+-ATPase with the transmembrane Vo-sector. Biochemistry 43, 12297-12305. DOI: 10.1021/bi0493867
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