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Proteins destined for the inner nuclear membrane (INM) start out in the peripheral ER. Diffusion from the ER will get them to the contiguous outer nuclear membrane (ONM), but the next step could involve either vesicular transport, short-lived fusions between INM and ONM, or movement along the lipid bilayers surrounding nuclear pores. On page 1051, Ohba et al. suggest that the last explanation applies. Moreover, their data suggest that the process can only occur because the nuclear pore complex, which was thought to be a static structure, is constitutively remodeled.- a! `8 @9 @& \) {# B, o
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Previous models suggested that INM proteins move through the nuclear pore membrane, but researchers needed a dynamic assay to test the model. By adapting a protein trapping system (Chen et al. 1995. Proc. Natl. Acad. Sci. USA. 92:4947–4951), Ohba et al. were able to watch fluorescently-labeled FRB reporter proteins move between the peripheral ER and the INM. Under normal conditions, the reporter moved between membranes without restriction. However, when the team added rapamycin to the cells, the FRB reporter accumulated in the INM as the reporter bound to an FK binding protein (FKBP) associated with the nuclear lamina. Thus, the rapamycin-mediated interaction trapped the reporter protein in the INM. The team thinks that native INM proteins become similarly trapped when they associate with nuclear structures./ Z! m/ `% a+ I. g0 W' \7 j5 v7 ]
, p/ s3 R$ v- q% IOnly proteins whose luminal and cytosolic domains were under 60 kD gained access to the INM, a limitation noted previously. INM localization was dependent on both energy and temperature, which would be consistent with membrane fusion events. However, because the addition of inhibitors of membrane fusion had no effect on localization in the INM, the team hypothesizes that the energy is required for nuclear pore remodeling. Indeed, addition of antibodies against the nuclear pore protein gp210 blocked localization to the INM.
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: a$ q3 Q: M% ~% i$ n: M) x# ?/ q5 ZWhile the pore structure is busy with the remodeling, small integral membrane proteins may slip by, passing into the INM. The team thinks pore remodeling is constitutive because reporter proteins that lacked any native nuclear protein sequence accumulated in the INM in the presence of rapamycin, suggesting that a signal is not required.(A reporter (green) gets trapped (bottom)) |
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发表于 2009-3-6 08:26
发表于 2015-5-31 16:52
