Isoform-specific differences in rapid nucleocytoplasmic shuttling cause distinct subcellular distributions of 14-3-3 sigma and 14-3-3 zeta.
Nucleocytoplasmic transport of proteins plays an important role in the regulation of many cellular processes. Differences in nucleocytoplasmic shuttling can provide a basis for isoform-specific biological functions for members of multigene families, like the 14-3-3 protein family. Many organisms contain multiple 14-3-3 isoforms, which play a role in numerous processes, including signalling, cell cycle control and apoptosis. It is still unclear whether these isoforms have specialised biological functions and whether this specialisation is based on isoform-specific ligand binding, expression regulation or specific localisation. Therefore, we studied the subcellular distribution of 14-3-3 sigma and 14-3-3 zeta in vivo in various mammalian cell types using yellow fluorescent protein fusions and isoform-specific antibodies. 14-3-3 sigma was mainly localised in the cytoplasm and only low levels were present in the nucleus, whereas 14-3-3 zeta was found at relatively higher levels in the nucleus. Fluorescence recovery after photobleaching (FRAP) experiments indicated that the 14-3-3 proteins rapidly shuttle in and out of the nucleus through active transport and that the distinct subcellular distributions of 14-3-3 sigma and 14-3-3 zeta are caused by differences in nuclear export. 14-3-3 sigma had a 1.7x higher nuclear export rate constant than 14-3-3 zeta, while import rate constants were equal. The 14-3-3 proteins are exported from the nucleus at least in part by a Crm1-dependent, leptomycin B-sensitive mechanism. The differences in subcellular distribution of 14-3-3 that we found in this study are likely to reflect a molecular basis for isoform-specific biological specialisation.
Section Molecular Cell Biology, Institute of Biology, Leiden University, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. hermert@rulbim.leidenuniv.nl
Martijn J
MJ
van Hemert
Maarten
M
Niemantsverdriet
Thomas
T
Schmidt
Claude
C
Backendorf
Herman P
HP
Spaink
D048948:14-3-3 Proteins / Q000737:chemistry*; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D020033:Protein Isoforms / Q000737:chemistry* / Q000378:metabolism*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry
D048948:14-3-3 Proteins; D005456:Fluorescent Dyes; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins