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 HIVA Tat protein responsible for cellular internalization. This Tat peptide was labelled at a C-terminal cysteine residue with the fluorescent probes IAF (5-lodoacetamidofluorescein) or A568 (Alexa Fluor 568). The Tat-IAF conjugate was directly observed entering liposomes at room temperature (approx. 25 degrees C) 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.