Translocation of the cell-penetrating Tat peptide across artificial bilayers and into living cells.
The ability of a short, charged peptide to penetrate synthetic DOPC (1,2-dioleoyl-sn-3-glycerophosphocholine) liposomes was investigated by fluorescence confocal microscopy. The peptide, termed Tat (trans-activating transcription factor), was a 14-mer derived from the region of the HIV-1 Tat protein responsible for cellular internalization. This Tat peptide was labelled at a C-terminal cysteine residue with the fluorescent probes IAF (5-iodoacetamidofluorescein) or A568 (Alexa Fluor 568). The Tat-IAF conjugate was directly observed entering liposomes at room temperature (approx. 258C) in the absence of pH gradient, ATP or other energy source. The uptake of the Tat-A568 conjugate in unfixed, live HeLa cells was found to be via endocytosis, as expected. In contrast, when the peptide was attached to an IAF-labelled 25 kDa protein corresponding to the catalytic domain of Clostridium botulinum C3 exotoxin, this larger, Tat-C3-IAF construct was not able to enter liposomes, although it localized similarly to Tat-A568 in live cells. The data suggest that Tat peptide can cross synthetic bilayers spontaneously in vitro, but that size and type of cargo may limit this behaviour.
Curnow
Paul
P
Department of Biochemistry, University of Bristol, University Walk, Bristol BS8 1TD, U.K. Curnow@lifesci.ucsb.edu
Mellor
Harry
H
Stephens
David J
DJ
Lorch
Mark
M
Booth
Paula J
PJ
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D001905:Botulinum Toxins / Q000737:chemistry / Q000378:metabolism; D004705:Endocytosis; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015696:Gene Products, tat / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism; D054322:tat Gene Products, Human Immunodeficiency Virus
D005452:Fluoresceins; D005456:Fluorescent Dyes; D015696:Gene Products, tat; D008051:Lipid Bilayers; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D054322:tat Gene Products, Human Immunodeficiency Virus; C422951:tat peptide (48-60), Human immunodeficiency virus 1; C015296:5-iodoacetamidofluorescein; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D001905:Botulinum Toxins; C017251:1,2-oleoylphosphatidylcholine