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J. Biol. Chem., Vol. 278, Issue 50, 50188-50194, December 12, 2003

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Cytoplasmic and Nuclear Delivery of a TAT-derived Peptide and a -Peptide after Endocytic Uptake into HeLa Cells^*

Terra B. Potocky, Anant K. Menon¶, and Samuel H. Gellman||

From the Departments of Chemistry and Biochemistry, University of Wisconsin, Madison, Wisconsin 53706

Several short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports have questioned this conclusion, attributing the prior observations to artifacts resulting from fixation procedures used to prepare cells for fluorescence microscopy. These studies analyzed live cells and showed that the peptides entered through endocytosis and accumulated in endocytic vesicles, without necessarily entering the cytoplasm. To resolve this controversy and to extend the analyses to non-natural -peptide sequences, we studied the cytoplasmic and nuclear delivery of a fluorescein-labeled 9-residue sequence derived from the human immunodeficiency virus transactivator of transcription (TAT) peptide, TAT-(47–57), as well as a similarly labeled 12-residue -peptide, -(VRR)₄, in live cells. Using fluorescence confocal microscopy, we show that when added to cells, both peptides are found in endocytic vesicles containing the transferrin receptor as well as in the cytoplasm and nucleus (TAT-(47–57)) or nucleolus (-(VRR)₄). The cells were verified to be intact through all experimental procedures by demonstrating their ability to exclude propidium iodide. Endocytic entry of the peptides was blocked by the energy poisons sodium azide and 2-deoxyglucose, whereas staining of the nucleus (nucleolus), but not endocytic vesicles, was abrogated by treating the cells with ammonium chloride. Our observations are consistent with the proposal that TAT-(47–57) and -(VRR)₄ enter cells by endocytosis and then exit an endosomal compartment to enter the cytoplasm by means of a mechanism requiring endosome acidification.

Received for publication, August 7, 2003 , and in revised form, September 24, 2003.

^* This work was supported by National Institutes of Health Grants GM56414 (to S. H. G.) and GM55427 (to A. K. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Dept. of Biochemistry, Univ. of Wisconsin, 433 Babcock Dr., Madison, WI 53706. Tel.: 608-262-2913; Fax: 608-262-3453; E-mail: menon{at}biochem.wisc.edu. ||To whom correspondence should be addressed: Dept. of Chemistry, Univ. of Wisconsin, 1101 University Ave., Madison, WI 53706. Tel.: 608-262-3303; Fax: 608-265-4534; E-mail: gellman{at}chem.wisc.edu.

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