Quantitative Analysis of Tat Peptide Binding to Import Carriers Reveals Unconventional Nuclear Transport Properties

A detailed study of nuclear import mediated by the HIV-1 Tat peptide ((47)YGRKKRRQRRR(57), Tat(RRR)) is reported. Fluorescence-based measurements, calibration of protein concentrations, and binding assays are exploited to address the physicochemical mechanisms of Tat peptide recognition by the classical importin alpha (Imp alpha) and importin beta (Imp beta) receptors both in vitro and in intact cells. We show that TatRRR is an unconventional nuclear localization sequence that binds directly to both Imp alpha and Imp beta carriers in the absence of competitors (in vitro), whereas this property is silenced in the actual cellular environment. In the latter case, Imp alpha/beta-dependent nuclear import can be successfully restored by replacing the "RRR" stretch with "GGG". We apply a recently developed method to determine quantitatively Tat(GGG) affinity for each receptor. Based on these results, we can rationalize previous controversial reports on the Tat peptide and provide coherent guidelines for the design of novel intracellular targeting sequences.