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Title: An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium

Abstract

Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD ({sup 171}EDVSRFIKGKLLQKQQKIYKDLERF{sup 195}) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues {sup 48}KKSYQDPEIIAHSRPRK{sup 64} that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to {sup 48}EF{sup 49} abolished the stability of the GFP fusionmore » at the protein but not RNA level preventing subcellular analyses. Cells transfected with the {sup 48}EF{sup 49} construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium.« less

Authors:
 [1];  [1]
  1. Department of Biology, University of Toronto at Mississauga, Mississauga, Ont. (Canada)
Publication Date:
OSTI Identifier:
20710818
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 332; Journal Issue: 1; Other Information: DOI: 10.1016/j.bbrc.2005.04.101; PII: S0006-291X(05)00857-0; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AMINO ACIDS; CALMODULIN; COMPUTER-AIDED MANUFACTURING; MUTANTS; MUTATIONS; NUCLEI; RESIDUES; RNA; TRANSLOCATION

Citation Formats

Myre, Michael A, and O'Day, Danton H. An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium. United States: N. p., 2005. Web. doi:10.1016/j.bbrc.2005.04.101.
Myre, Michael A, & O'Day, Danton H. An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium. United States. doi:10.1016/j.bbrc.2005.04.101.
Myre, Michael A, and O'Day, Danton H. Fri . "An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium". United States. doi:10.1016/j.bbrc.2005.04.101.
@article{osti_20710818,
title = {An N-terminal nuclear localization sequence but not the calmodulin-binding domain mediates nuclear localization of nucleomorphin, a protein that regulates nuclear number in Dictyostelium},
author = {Myre, Michael A and O'Day, Danton H},
abstractNote = {Nucleomorphin is a novel nuclear calmodulin (CaM)-binding protein (CaMBP) containing an extensive DEED (glu/asp repeat) domain that regulates nuclear number. GFP-constructs of the 38 kDa NumA1 isoform localize as intranuclear patches adjacent to the inner nuclear membrane. The translocation of CaMBPs into nuclei has previously been shown by others to be mediated by both classic nuclear localization sequences (NLSs) and CaM-binding domains (CaMBDs). Here we show that NumA1 possesses a CaMBD ({sup 171}EDVSRFIKGKLLQKQQKIYKDLERF{sup 195}) containing both calcium-dependent-binding motifs and an IQ-like motif for calcium-independent binding. GFP-constructs containing only NumA1 residues 1-129, lacking the DEED and CaMBDs, still localized as patches at the internal periphery of nuclei thus ruling out a direct role for the CaMBD in nuclear import. These constructs contained the amino acid residues {sup 48}KKSYQDPEIIAHSRPRK{sup 64} that include both a putative bipartite and classical NLS. GFP-bipartite NLS constructs localized uniformly within nuclei but not as patches. As with previous work, removal of the DEED domain resulted in highly multinucleate cells. However as shown here, multinuclearity only occurred when the NLS was present allowing the protein to enter nuclei. Site-directed mutation analysis in which the NLS was changed to {sup 48}EF{sup 49} abolished the stability of the GFP fusion at the protein but not RNA level preventing subcellular analyses. Cells transfected with the {sup 48}EF{sup 49} construct exhibited slowed growth when compared to parental AX3 cells and other GFP-NumA1 deletion mutants. In addition to identifying an NLS that is sufficient for nuclear translocation of nucleomorphin and ruling out CaM-binding in this event, this work shows that the nuclear localization of NumA1 is crucial to its ability to regulate nuclear number in Dictyostelium.},
doi = {10.1016/j.bbrc.2005.04.101},
journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 1,
volume = 332,
place = {United States},
year = {2005},
month = {6}
}