title: Quantitative analysis of Tat peptide binding to import carriers reveals unconventional nuclear transport properties.
abstract: A detailed study of nuclear import mediated by the HIV-1 Tat peptide (47YGRKKRRQRRR57, TatRRR) 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 α (Impα) and importin β (Impβ) receptors both in vitro and in intact cells. We show that TatRRR is an unconventional nuclear localization sequence that binds directly to both Impα and Impβ carriers in the absence of competitors (in vitro), whereas this property is silenced in the actual cellular environment. In the latter case, Impα/β-dependent nuclear import can be successfully restored by replacing the "RRR" stretch with "GGG". We apply a recently developed method to determine quantitatively TatGGG 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.
authors.affiliation: Center for Nanotechnology Innovation @NEST, Istituto Italiano di TecnologiaConsiglio Nazionale delle Ricerche, Scuola Normale Superiore, Pisa, Italy. francesco.cardarelli@iit.it
authors.forename: Francesco
authors.initials: F
authors.lastname: Cardarelli
authors.forename: Michela
authors.initials: M
authors.lastname: Serresi
authors.forename: Alberto
authors.initials: A
authors.lastname: Albanese
authors.forename: Ranieri
authors.initials: R
authors.lastname: Bizzarri
authors.forename: Fabio
authors.initials: F
authors.lastname: Beltram
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000235:genetics / Q000502:physiology*; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D036681:Fluorescence Recovery After Photobleaching; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D028901:alpha Karyopherins / Q000235:genetics / Q000378:metabolism*; D028961:beta Karyopherins / Q000235:genetics / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000378:metabolism*
chemical_list: D010446:Peptide Fragments; D028901:alpha Karyopherins; D028961:beta Karyopherins; D054322:tat Gene Products, Human Immunodeficiency Virus