TAT Fusion Protein Transduction into Isolated Mitochondria Is Accelerated by Sodium Channel Inhibitors

Abstract

Stringent control of ion and protein transport across the mitochondrial membranes is required to maintain mitochondrial function and biogenesis. In particular, the inner mitochondrial membrane is generally impermeable to proteins entering the matrix except via tightly regulated protein import mechanisms. Recently, cell penetrant peptides have been shown to move across the inner mitochondrial membrane in a manner suggesting an independent mechanism. HIV-1 transactivator of transcription (TAT) is an arginine-rich cell penetrant peptide, 47YGRKKRRQRRR57, which can transduce full-length proteins not only across the cell membrane but also into intracellular organelles. In this study, we investigated the ability of a TAT-containing protein to move into the mitochondrial matrix. Using a novel FACS assay for isolated, purified mitochondria, we show that TAT can deliver a modified fluorescent protein, mMDH-GFP, to the matrix of mitochondria and it is subsequently processed by the matrix peptidases. In addition, transduction of TAT-mMDH-GFP into mitochondria is independent of canonical protein import pathways as well as mitochondrial membrane potential. In direct contrast to published reports regarding the cell membrane where the sodium channel inhibitor, amiloride, blocks endocytosis and inhibits TAT transduction, TAT transduction into mitochondria is markedly increased by this same sodium channel inhibitor. These results confirm that the cell penetrant peptide, TAT, can readily transduce a protein cargo into the mitochondrial matrix. These results also demonstrate a novel role for mitochondrial sodium channels in mediating TAT transduction into mitochondria that is independent of endocytotic mechanisms. The mechanism of TAT transduction into mitochondria therefore is distinctly different from transduction across the cell membrane.

Downregulation of doxorubicin-induced myocardial apoptosis accompanies postnatal heart maturation

“…Heart mitochondria were freshly isolated on the day of the experiments using the previously described methods (20). Briefly, hearts were homogenized in ice-cold mitochondrial isolation buffer (20 mM HEPES pH 7.4, 190 mM mannitol, and 70 mM sucrose) and then subjected to three rounds of differential centrifugation at 700 g, 900 g, and 1,000 g at 4°C for 10 min to remove cell debris and nuclei.…”

Replacement of the C6ORF66 Assembly Factor (NDUFAF4) Restores Complex I Activity in Patient Cells

“…PTDs, such as TAT, have the ability to penetrate the plasma membrane as well as the mitochondrial membranes (26,35,36), leading to the possible application of the PTD technology to a protein-therapeutic approach for mitochondrial disorders. Besides our research regarding the TAT-LAD fusion protein (see the Introduction), a number of studies have been reported.…”

Transduction of Human Recombinant Proteins into Mitochondria as a Protein Therapeutic Approach for Mitochondrial Disorders

“…into pregnant mice, was detected in mitochondria of both maternal and fetal organs, like heart and brain. A novel mechanism for TAT transduction of mitochondrial membranes through the mitochondrial sodium channels has been proposed (173), different from the endocytotic mechanisms proposed for TAT transduction of plasma membranes.…”

“…The ability of PTDs (like TAT) to penetrate not only the plasma membrane but also the mitochondrial membranes (154,172,173) "opened the door" for the application of PTD technology as a putative protein therapeutic approach of mitochondrial disorders. In general, TAT could mediate transduction of the PT either from its N-terminal (TAT-X fusion protein) or its C-terminal (X-TAT fusion protein).…”