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Vol. 273, Issue 3, 1623-1628, January 16, 1998
The HIV-1 Tat Nuclear Localization Sequence Confers Novel Nuclear
Import Properties
Athina
Efthymiadis,
Lyndall J.
Briggs, and
David A.
Jans
From the Nuclear Signaling Laboratory, Division of Biochemistry and
Molecular Biology, John Curtin School of Medical Research, Canberra,
A.C.T. 2601, Australia
The different classes of conventional
nuclear localization sequences (NLSs) resemble one another in that
NLS-dependent nuclear protein import is
energy-dependent and mediated by the cytosolic NLS-binding
importin/karyopherin subunits and monomeric GTP-binding protein
Ran/TC4. Based on analysis of the nuclear import kinetics mediated by
the NLS of the human immunodeficiency virus accessory protein Tat using
in vivo and in vitro nuclear transport assays and confocal laser scanning microscopy, we report a novel nuclear import pathway. We demonstrate that the Tat-NLS, not recognized by
importin 58/97 subunits as shown using an enzyme-linked immunosorbent assay-based binding assay, is sufficient to target the 476-kDa heterologous -galactosidase protein to the nucleus in
ATP-dependent but cytosolic factor-independent fashion.
Excess SV40 large tumor antigen (T-ag) NLS-containing peptide had no
significant effect on the nuclear import kinetics implying that the
Tat-NLS was able to confer nuclear accumulation through a pathway
distinct from conventional NLS-dependent pathways.
Nucleoplasmic accumulation of the Tat-NLS- -galactosidase fusion
protein, in contrast to that of a T-ag-NLS-containing fusion protein,
also occurred in the absence of an intact nuclear envelope, implying
that the Tat-NLS conferred binding to nuclear components. This is in
stark contrast to known NLSs such as those of T-ag which confer nuclear
entry rather than retention. Significantly, the ability to accumulate in the nucleus in the absence of an intact nuclear envelope was blocked
in the absence of ATP, as well as by nonhydrolyzable ATP and GTP
analogs, demonstrating that ATP is required to effect release from a
complex with insoluble cytoplasmic components. Taken together, the
results demonstrate that, dependent on ATP for release from cytoplasmic
retention, the Tat-NLS is able to confer nuclear entry and binding to
nuclear components. These unique properties indicate that Tat
accumulates in the nucleus through a novel import pathway.
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.
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