Lipophilic triphenylphosphonium cations as tools in mitochondrial bioenergetics and free radical biology.

Lipophilic phosphonium cations were first used to investigate mitochondrial biology by Vladimir Skulachev and colleagues in the late 1960s. Since then, these molecules have become important tools for exploring mitochondrial bioenergetics and free radical biology. Here we review why these molecules are useful in mitochondrial research and outline some of the ways in which they are now being utilized.

Cell-penetrating peptides are excluded from the mitochondrial matrix.

CPPs (cell-penetrating peptides) facilitate cellular uptake of covalently attached macromolecules, through an as yet controversial mechanism that either involves direct membrane passage or a type of endocytosis. We investigated the potential of the CPPs penetratin and Tat to act as mitochondria-targeting vectors by testing whether they were internalized by isolated mitochondria, and by mitochondria within cells in culture. We also tested peptides conjugated to the mitochondria-targeting moiety triphenylphosphonium. We found no evidence for mitochondrial uptake by penetratin, Tat or their triphenylphosphonium conjugates. This result suggests that CPPs are unsuitable as mitochondria-targeting vectors, and implies an endocytic mode of cellular uptake for CPPs.

Using mitochondria-targeted molecules to study mitochondrial radical production and its consequences.

The production of ROS (reactive oxygen species) by the mitochondrial respiratory chain contributes to a range of pathologies, including neurodegenerative diseases, ischaemia/reperfusion injury and aging. There are also indications that mitochondrial ROS production plays a role in damage response and signal transduction pathways. To unravel the role of mitochondrial ROS production in these processes, we have developed a range of mitochondria-targeted probe molecules. Covalent attachment of a lipophilic cation leads to their accumulation into mitochondria, driven by the membrane potential. Molecules developed so far include antioxidants designed to intercept mitochondrial ROS and reagents that specifically label mitochondrial thiol proteins. Here we outline how mitochondrial ROS formation and its consequences can be investigated using these probes.