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Nuclear import
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Nuclear import Hicks, G.R., Smith, H.M.S., Lobreaux, S. and Raikhel, N.V. (1996) Nuclear import in permeabilized protoplasts from higher plants has unique features, Plant Cell 8, 1337-1352 Pores in the nuclear envelope allow the passage of macromolecules into and out of the nucleus. The translocation of proteins through the pore complex requires a nuclear localization sequence (NLS), of which three general classes have been identified in plants and other eukaryotes. Previous research in Raikhel's lab has shown that proteins containing an NLS from plant or animal sources can bind to the outside of isolated plant nuclei. They have also demonstrated that the introduction of a plasmid containing a ~-glucuronidase gene combined with a plant NLS results in nuclear localization of the protein in vivo. In the present paper, they have used a combination of these two approaches to show nuclear import of protein in vitro. A new permeabilizing system has been developed, which - in contrast to the use of detergents such as Triton-Xl00 - leaves the nuclear envelope intact. It has thus been possible to assess the translocation of fluorescently tagged human serum albumin coupled to a plant NLS. Unlike the results for similar assays using animal cells, no additional cytosol was required for import. This prompted the search for 'essential import factors' that might remain in association with the permeabilized protoplasts. A partial cDNA (from the Arabidopsis expressed sequence tag project) with homology to yeast and animal importin a sequences was used to identify a full-length clone in Arabidopsis. Antibodies raised to this cross-reacted with a tobacco homologue and mammalian importin ~, and revealed that plant importin a receptors are retained in permeabilized protoplasts a particularly strong signal was detected in the vicinity of the nucleus. Other interesting differences in import between animals and plants include that: it can occur at lower temperatures in plants; and that it is inhibited by the addition of non-hydrolysable GTP, but not ATP analogues, whereas in animal systems both ATP and GTP are required. The results obtained using this novel system pose many questions about the mechanics of plant nuclear import and how it may differ from animal and yeast models - but they are questions that can now be addressed.
370
November1996, Vol. 1, No. 11
Copyright © 1996 Elsevier Science Ltd. All rights reserved. 1360 - 1385/96/$15.00
news
Nuclear import Hicks, G.R., Smith, H.M.S., Lobreaux, S. and Raikhel, N.V. (1996) Nuclear import in permeabilized protoplasts from higher plants has unique features, Plant Cell 8, 1337-1352 Pores in the nuclear envelope allow the passage of macromolecules into and out of the nucleus. The translocation of proteins through the pore complex requires a nuclear localization sequence (NLS), of which three general classes have been identified in plants and other eukaryotes. Previous research in Raikhel's lab has shown that proteins containing an NLS from plant or animal sources can bind to the outside of isolated plant nuclei. They have also demonstrated that the introduction of a plasmid containing a ~-glucuronidase gene combined with a plant NLS results in nuclear localization of the protein in vivo. In the present paper, they have used a combination of these two approaches to show nuclear import of protein in vitro. A new permeabilizing system has been developed, which - in contrast to the use of detergents such as Triton-Xl00 - leaves the nuclear envelope intact. It has thus been possible to assess the translocation of fluorescently tagged human serum albumin coupled to a plant NLS. Unlike the results for similar assays using animal cells, no additional cytosol was required for import. This prompted the search for 'essential import factors' that might remain in association with the permeabilized protoplasts. A partial cDNA (from the Arabidopsis expressed sequence tag project) with homology to yeast and animal importin a sequences was used to identify a full-length clone in Arabidopsis. Antibodies raised to this cross-reacted with a tobacco homologue and mammalian importin ~, and revealed that plant importin a receptors are retained in permeabilized protoplasts a particularly strong signal was detected in the vicinity of the nucleus. Other interesting differences in import between animals and plants include that: it can occur at lower temperatures in plants; and that it is inhibited by the addition of non-hydrolysable GTP, but not ATP analogues, whereas in animal systems both ATP and GTP are required. The results obtained using this novel system pose many questions about the mechanics of plant nuclear import and how it may differ from animal and yeast models - but they are questions that can now be addressed.
370
November1996, Vol. 1, No. 11
Copyright © 1996 Elsevier Science Ltd. All rights reserved. 1360 - 1385/96/$15.00