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- Title
Characterization of RanBPM sequence components that regulate its subcellular localization.
- Authors
Dale, Robert; Atabakhsh, Elnaz; Fell, Victoria; Schild-Poulter, Caroline
- Abstract
Ran-binding protein M (RanBPM) is a 90 kDa nucleocytoplasmic protein that has been implicated as a pro-apoptotic factor. In addition, it has been found to translocate out of the nucleus and into the cytoplasm following ionizing radiation (IR) treatment. Thus, RanBPM's subcellular localization may play a role in regulating its pro-apoptotic function. Since RanBPM is over the 50 kDa passive diffusion size limit for a nuclear pore complex, it must be actively transported across the nuclear membrane. The purpose of this study was to determine which regions of RanBPM are responsible for regulating its subcellular localization. HeLa cells stably expressing siRNA targeted against endogenous RanBPM were transfected with RanBPM constructs containing various point mutations and deletions. Constructs also contained point mutations in the C-terminal to make them resistant to siRNA-mediated knockdown. An HA tag was fused to each construct to enable visualization using indirect immunofluorescence microscopy. The N-terminal region (residues 1-251) was found to be important for subcellular localization. Specifically, residues 1-101 were found to promote nuclear retention whereas the N-terminal putative NES was found to facilitate nuclear export. In addition, deletion of RanBPM's SPRY domain localized RanBPM to the cytoplasm and caused significant aggregation. This suggest the SPRY domain may be important for cytoplasmic maintenance and solubility.
- Subjects
CARRIER proteins; APOPTOSIS; CYTOPLASM; IMMUNOLOGY technique; CELL aggregation
- Publication
UBC Medical Journal, 2011, Vol 2, Issue 2, p29
- ISSN
1920-7425
- Publication type
Abstract