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Michael Duchen & Richard Youle Collaboration

Title: THE CELL BIOLOGY OF THE PROTEIN IF1: Theimpact of mitochondrial proteinIF1 on metabolism, cell physiology and roles in cell death and autophagy in the CNS

UCL Supervisor: Michael R Duchen, Ph.D., email: m.duchen@ucl.ac.uk  
NIH Supervisor: Richard Youle, Ph.D., email: YouleR@ninds.nih.gov

Introduction

The F1F0-ATPsynthase operates as a molecular motor which synthesises ATP in a process driven by the proton gradient and potential across the mitochondrial membrane. When that proton gradient is compromised (e.g. during hypoxia) the enzyme runs ‘backwards’, acting as an ATP dependent proton pump, consuming glycolytic ATP in a process that sustains the mitochondrial membrane potential. In cells with a high mitochondrial density, mitochondria in which function has been compromised can paradoxically CONSUME all cellular ATP reserves in minutes. IF1, the inhibitor of the F1 mitochondrial subunit, is a small intrinsic protein which regulates the activity of the mitochondrial ATPase in its ‘reverse mode’, binding to the ATPase and inhibiting this consumption.

We have shown that overexpression of the protein modulates mitochondrial function and is protective against hypoxic injury. We have discovered that it also modulates mitochondrial ATP synthesis, that it alters mitochondrial cristae structure, and also that it is protective against both necrotic and apoptotic cell death. Its expression levels vary hugely between cell types and tissues - we have no idea why. It is also upregulated in a number of cancers. We expect soon to have transgenic animals conditionally overexpressing the protein in neurons. Specific neuroscience interests in IF1 include: exploring variations in expression levels in different areas of the CNS; exploring the relative expression levels in different cell types; examining the functional consequences of expression for cell death to hypoxia and reperfusion, to glutamate toxicity and to Amyloid toxicity.

Methods

Methods include standard molecular biological manipulations to overexpress or knockout the protein in cells and tissues, using immunofluorescence and Western Blots to explore expression levels, live cell confocal imaging to follow changes in potential, calcium and free radical generation in cells and tissues, and using GFP tagged proteins (e.g. cyt c/bax ) to explore translocation of proteins during apoptosis.

References

Campanella M, Casswell E, Chong S, Farah Z, Wieckowski MR, Abramov AY, Tinker A, Duchen MR. Regulation of mitochondrial structure and function by the F1Fo-ATPase inhibitor protein, IF1. Cell Metab. 2008 Jul;8(1):13-25.

Campanella M, Parker N, Tan CH, Hall AM, Duchen MR. IF(1): setting the pace of the F(1)F(o)-ATP synthase. Trends Biochem Sci. 2009 Jul;34(7):343-50

Suen DF, Norris KL, Youle RJ. Mitochondrial dynamics and apoptosis. Genes Dev. 2008 Jun 15;22(12):1577-90.