A novel TAT-mitochondrial signal sequence fusion protein is processed, stays in mitochondria, and crosses the placenta.
Mutations in nuclear and mitochondrial genomes can lead to defects in mitochondrial function. To date, repair of these defects with exogenous proteins or gene transfer has been difficult with either viral or nonviral vectors. We hypothesized that TAT fusion proteins would cross both mitochondrial membranes and that incorporation of a mitochondrial signal sequence into a TAT fusion protein would allow processing and localization of exogenous proteins in mitochondria. A TAT-mitochondrial malate dehydrogenase signal sequence (mMDH)-enhanced green fluorescent protein (eGFP) fusion protein was constructed. TAT-mMDH-eGFP allowed rapid transduction and localization of fusion protein into mitochondria of multiple cell types. In contrast, TAT-GFP, without a mitochondrial signal sequence, rapidly transduced into cells and mitochondria, displayed pseudo-first-order kinetics, but did not remain there. Mice injected 5 days prior with TAT-mMDH-eGFP had detectable eGFP activity in multiple tissue types. Western blotting of cytosolic and mitochondrial fractions isolated from their livers confirmed eGFP localization to mitochondria and that the mMDH transit peptide was recognized and processed. Furthermore, TAT-mMDH-eGFP fusion protein injected into pregnant mice crossed the placenta and was detectable in both the fetus and the newborn pups. TAT fusion proteins containing a mitochondrial signal sequence are a viable method to localize proteins to mitochondria.
Del Gaizo
Victoria
V
Division of Molecular Medicine, Department of Internal Medicine, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157-1081, USA.
Payne
R Mark
RM
D000818:Animals; D005260:Female; D015696:Gene Products, tat / Q000235:genetics / Q000378:metabolism*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D007231:Infant, Newborn; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008930:Mitochondria, Liver / Q000378:metabolism*; D041681:NIH 3T3 Cells; D016716:PC12 Cells; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage / Q000378:metabolism*; D014161:Transduction, Genetic; D014162:Transfection; D054322:tat Gene Products, Human Immunodeficiency Virus
D015696:Gene Products, tat; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D008291:Malate Dehydrogenase