The uptake of HIV Tat peptide proceeds via two pathways which differ from macropinocytosis

•CPP Tat-peptide membrane translocation is an immediate, temperature-independent process.•CPP Tat-peptide uptake has linear temperature dependence possessing a low Q10 value.•CPP Tat-peptide does not enter the cells in the same endocytosis route as dextran.

Cell penetrating peptides (CPPs) have been extensively studied as vectors for cellular delivery of therapeutic molecules, yet the identity of their uptake routes remained unclear and is still under debate. In this study we provide new insights into CPP entry routes by quantitatively measuring the intracellular uptake of FAM-labeled Tat-peptide under rigorous kinetic and thermal conditions. The uptake of Tat-peptide between 4 and 15 °C corresponds to Q10 = 1.1, proceeding through a prompt (< 5 min), temperature-independent process, suggesting direct membrane translocation. At longer durations, Tat rate of uptake shows linear dependence on temperature with Q10 = 1.44, accompanied by activation energy Ea = 4.45 Kcal/mole. These values are significantly lower than those we found for the macropinocytosis probe dextran (Q10 = 2.2 and Ea = 7.2 Kcal/mole) which possesses an exponential dependence on temperature, characteristic of endocytosis processes. Tat-peptide and dextran do not interfere with each other's uptake rate and the ratio of Tat-peptide uptake to its extracellular concentration is ~ 15 times higher than that for dextran. In addition, Phloretin, a modulator of cell membrane dipole potential, is shown to increase dextran uptake but to reduce that of Tat. We conclude that the uptake of Tat differs from that of dextran in all parameters. Tat uptake proceeds by dual entry routes which differ by their energy dependence.

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