Targeting proteins to mitochondria using TAT.
Disorders of mitochondrial function cause significant human disease and suffering. To date, correction of these mitochondria defects has depended on biochemical approaches and has not been achieved via gene therapies. Using previously described fusion proteins containing the transactivator of transcription (TAT) region from the HIV virus and green fluorescent protein (GFP), with and without a mitochondrial targeting sequence (MTS) from mitochondrial malate dehydrogenase (mMDH), we have investigated transduction across mitochondrial membranes. Both TAT-GFP and TAT-mMDH-GFP fusion proteins are protected from externally added protease when incubated with isolated mitochondria. Furthermore, both TAT fusion proteins rapidly enter cultured cells and transduce into mitochondria. However, the MTS allows processing of the fusion protein and is necessary for persistence in mitochondria over time. Neither degradation of import receptors nor disruption of the mitochondrial membrane potential or pH gradient inhibits protein transduction of either fusion protein. Furthermore, when injected into pregnant mice, TAT-mMDH-GFP is detectable throughout fetal and neonatal pups. These results indicate that TAT fusion proteins are able to traverse mitochondrial membranes through mechanisms that do not involve the regular import pathway, and that the addition of a MTS allows persistence of the fusion protein within mitochondria. TAT-MTS fusion proteins may represent a viable option as potential mitochondrial protein therapies.
Division of Molecular Medicine, Department of Internal Medicine, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, NC 27157-1081, USA.
Victoria
V
Del Gaizo
James A
JA
MacKenzie
R Mark
RM
Payne
D000818:Animals; D005260:Female; D005333:Fetus / Q000737:chemistry / Q000166:cytology; D015696:Gene Products, tat / Q000235:genetics*; D049452:Green Fluorescent Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000737:chemistry / Q000166:cytology; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008168:Lung / Q000737:chemistry / Q000166:cytology; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009206:Myocardium / Q000737:chemistry / Q000166:cytology; D016716:PC12 Cells; D011247:Pregnancy; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D015696:Gene Products, tat; D008164:Luminescent Proteins; D024101:Mitochondrial Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D008291:Malate Dehydrogenase