Regulation of nuclear import by light‐induced activation of caged nuclear localization signal in living cells

A novel fluorescence probe suitable for the study of nuclear import in living cells has been developed. The lysine‐128 residue in SV40 T‐antigen nuclear localization signal (NLS) was converted to a caged lysine with the amino acid blocked by a photocleavable protecting group. Following irradiation of ultraviolet (UV) light, the caged NLS conjugate translocated into and accumulated in the nucleus within 20 min similar to uncaged NLS conjugate. Maximum import rate saturated approximately 4.78±0.21% per minute when the duration of irradiation was more than 1/15 s (22 mW/cm2). Caged NLS conjugate tended to distribute near the surface of the nucleus, and this association became stronger after UV irradiation. The caged conjugate enabled us to regulate the initial state of the reaction, both spatially and temporally.

[1]  G. Blobel,et al.  Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[2]  M. Roederer,et al.  Cy7PE and Cy7APC: bright new probes for immunofluorescence. , 1996, Cytometry.

[3]  G. Marriott.  Caged protein conjugates and light-directed generation of protein activity: preparation, photoactivation, and spectroscopic characterization of caged G-actin conjugates. , 1994, Biochemistry.

[4]  A. Dickmanns,et al.  Nucleocytoplasmic Shuttling Factors Including Ran and CRM1 Mediate Nuclear Export of NFAT In Vitro , 1998, The Journal of cell biology.

[5]  I. Cserpán,et al.  The mechanism of nuclear transport of natural or artificial transport substrates in digitonin-permeabilized cells. , 1995, Journal of cell science.

[6]  M. Moore,et al.  Getting across the nuclear pore complex. , 1999, Trends in cell biology.

[7]  S. Adam,et al.  Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors , 1990, The Journal of cell biology.

[8]  J. Moroianu.  Distinct nuclear import and export pathways mediated by members of the karyopherin β family , 1998, Journal of cellular biochemistry.

[9]  Y. Yoneda.  How proteins are transported from cytoplasm to the nucleus. , 1997, Journal of biochemistry.

[10]  S. R. Parker,et al.  Cyanine dye labeling reagents--carboxymethylindocyanine succinimidyl esters. , 1990, Cytometry.

[11]  Y. Yoneda,et al.  Up‐regulation of nuclear protein import by nuclear localization signal sequences in living cells , 1999, FEBS letters.

[12]  C. Dargemont,et al.  Nuclear transport and transcriptional regulation , 1999, FEBS letters.

[13]  J. Finch,et al.  Nuclear localization signal recognition causes release of importin-alpha from aggregates in the cytosol. , 1999, Journal of molecular biology.

[14]  S. Yoshikawa,et al.  Solid-phase synthesis of caged peptides using tyrosine modified with a photocleavable protecting group: application to the synthesis of caged neuropeptide Y. , 1996, Biochemical and biophysical research communications.

[15]  S. Adam,et al.  Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope , 1994, The Journal of cell biology.

[16]  D. Goldfarb,et al.  In vivo nuclear transport kinetics in Saccharomyces cerevisiae: a role for heat shock protein 70 during targeting and translocation , 1996, The Journal of cell biology.

[17]  Y. Shigeri,et al.  Synthesis of caged peptides using caged lysine: application to the synthesis of caged AIP, a highly specific inhibitor of calmodulin-dependent protein kinase II. , 1999, Bioorganic & medicinal chemistry letters.

[18]  S. R. Wente,et al.  The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm. , 2000, Current opinion in cell biology.

[19]  G. Schlenstedt.  Protein import into the nucleus , 1996, FEBS letters.

[20]  R. Laskey,et al.  A polypeptide domain that specifies migration of nucleoplasmin into the nucleus , 1982, Cell.

[21]  S. Adam,et al.  Sequence and characterization of cytoplasmic nuclear protein import factor p97 , 1995, The Journal of cell biology.

[22]  C. J. Lewis,et al.  Cyanine dye labeling reagents: sulfoindocyanine succinimidyl esters. , 1993, Bioconjugate chemistry.

[23]  G. Dreyfuss,et al.  A nuclear localization domain in the hnRNP A1 protein , 1995, The Journal of cell biology.

[24]  M. Hirano,et al.  Restricted photorelease of biologically active molecules near the plasma membrane. , 1998, Biochemical and biophysical research communications.

[25]  E. Kiseleva,et al.  The nuclear pore complex: mediator of translocation between nucleus and cytoplasm. , 2000, Journal of cell science.

[26]  E. Hartmann,et al.  Distinct functions for the two importin subunits in nuclear protein import , 1995, Nature.

[27]  Imamoto Naoko,et al.  The nuclear pore-targeting complex binds to nuclear pores after association with a karyophile. , 1995 .

[28]  C. Müller,et al.  Structure of importin-β bound to the IBB domain of importin-α , 1999, Nature.

[29]  H. Higashida,et al.  Slow Synaptic Responses and Modulation , 2000, Springer Japan.

[30]  G. Blobel,et al.  Crystallographic Analysis of the Recognition of a Nuclear Localization Signal by the Nuclear Import Factor Karyopherin α , 1998, Cell.

[31]  R. Kennedy,et al.  Induction of nuclear transport with a synthetic peptide homologous to the SV40 T antigen transport signal , 1986, Cell.

[32]  T. Boulikas,et al.  Nuclear localization signals (NLS). , 1993, Critical reviews in eukaryotic gene expression.

[33]  Marriott,et al.  Caged peptides and proteins: new probes to study polypeptide function in complex biological systems. , 1999, Trends in plant science.

[34]  J. Newport.  Nuclear reconstitution in vitro: Stages of assembly around protein-free DNA , 1987, Cell.

[35]  H. Bayley,et al.  Caged peptides and proteins by targeted chemical modification. , 1998, Methods in enzymology.

[36]  J. Hauber,et al.  Direct observation of nucleocytoplasmic transport by microinjection of GFP-tagged proteins in living cells. , 1999, BioTechniques.

[37]  Erich A. Nigg,et al.  Nucleocytoplasmic transport: signals, mechanisms and regulation , 1997, Nature.