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First published online 2 March 2004
doi: 10.1242/jcs.00990

Journal of Cell Science 117, 1411-1420 (2004)
Published by The Company of Biologists 2004
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Isoform-specific differences in rapid nucleocytoplasmic shuttling cause distinct subcellular distributions of 14-3-3{sigma} and 14-3-3{zeta}

Martijn J. van Hemert1,*, Maarten Niemantsverdriet2, Thomas Schmidt3, Claude Backendorf2 and Herman P. Spaink1

1 Section Molecular Cell Biology, Institute of Biology, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
2 Laboratory of Molecular Genetics, Leiden Institute of Chemistry, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
3 Physics of Life Processes, Leiden Institute of Physics, Leiden University, Niels Bohrweg 2, 2333 CA Leiden, The Netherlands



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  		Fig. 1. Recombinant human 14-3-3 proteins complement the lethal disruption of yeast 14-3-3 genes. (A) GG1306-derived yeast strains expressing the indicated 14-3-3 isoforms were transferred from glucose medium (left) to 5-FOA-containing medium (right). Strains can only grow on this medium when they are able to lose plasmid YCplac33[BMH2], illustrating the ability of the foreign 14-3-3 isoform to take over the function of the yeast 14-3-3 proteins. (B) The absence of plasmid Ycplac33[BMH2] after 5-FOA treatment was determined by PCR. This plasmid yields a 400 bp product, besides a 600 bp internal control product. Western blots on 5 µg (C) or 2.5 µg (D) total protein from strains after 5-FOA treatment with an antiserum against yeast 14-3-3 proteins (C) or against Pan 14-3-3 proteins (D). Lane b contains 20 ng purified Bmh2p.

 


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  		Fig. 2. Recombinant human 14-3-3 isoforms form dimers. (A) Coomassie-stained polyacryl-amide gel of recombinant human 14-3-3 isoforms purified from yeast. (B) Gel filtration analysis of 6xhis-14-3-3{sigma}-CFP.

 


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  		Fig. 3. (A) Subcellular distribution of 14-3-3{sigma}-YFP and 14-3-3{zeta}-YFP in the indicated mammalian cells. (B) HaCaT and Hela cells immunostained for endogenous 14-3-3{sigma} and 14-3-3{zeta}. (C) Quantitative analysis of the subcellular distribution of 14-3-3{sigma} (red) and 14-3-3{zeta} (blue). Solid bars represent the average nuclear/cytoplasmic (FN/FC) ratio obtained with YFP-labelled 14-3-3 isoforms. Hatched bars represent the quantitative analysis of immunolocalisations of endogenous 14-3-3{sigma} and {zeta}. Standard deviations are indicated (black lines).

 


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  		Fig. 4. Subcellular distribution of 14-3-3 after biochemical cell fractionation. HaCaT cells were fractionated into a cytosolic (C) and nuclear extract (N). Equal amounts of total protein from both extracts were analysed by western blotting. (A) Blot stained with specific 14-3-3{sigma} antibodies or (B) specific 14-3-3{zeta} antibodies. (C) Control probed with antibodies against known nuclear (p53), cytoplasmic (RasGAP, PKC{zeta}) and cytoskeletal (actin, {alpha}-tubulin) proteins.

 


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  		Fig. 5. Effect of LMB on intracellular 14-3-3{sigma} and 14-3-3{zeta} distribution. (A) HaCaT cells expressing 14-3-3{sigma}-YFP or 14-3-3{zeta}-YFP were incubated in medium with or without LMB for 5 hours. (B) Plot of the individual FN/FC ratios for 14-3-3{sigma}-YFP and 14-3-3{zeta}-YFP of 50 cells, with and without LMB treatment. (C) Percentage of cells with a significantly increased FN/FC ratio (FN/FC>0.45 for 14-3-3{sigma} and FN/FC>0.77 for 14-3-3{zeta}). (D) HaCaT cells incubated in medium with or without LMB for 5 hours were immunostained with specific antibodies for 14-3-3{sigma} or 14-3-3{zeta}. (E) Plot of the individual FN/FC ratios for endogenous 14-3-3{sigma} and 14-3-3{zeta} of cells treated with and without LMB. (F) Percentage of cells with a significantly increased FN/FC ratio of endogenous 14-3-3 (FN/FC>0.31 for 14-3-3{sigma} and FN/FC>0.64 for 14-3-3{zeta}).

 


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  		Fig. 6. FRAP analysis of nucleocytoplasmic transport of 14-3-3 proteins. (A) A region covering >75% of the nucleus (indicated by the white box) of HaCaT cells expressing 14-3-3{sigma}-YFP or 14-3-3{zeta}-YFP was photobleached, after which images were recorded at 1-second intervals. Fluorescence recovery is shown for two representative cells at the indicated times in seconds. (B) Typical normalised recovery curves with {tau}1/2 for nuclear export for 14-3-3{sigma}-YFP, 14-3-3{zeta}-YFP and YFP alone. (C) Rate constants for the nuclear import and export of 14-3-3{sigma}-YFP, 14-3-3{zeta}-YFP and YFP alone, in untreated and LMB-treated cells. At least 15 cells were measured and quantified in each experiment.

 

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