The molecular mechanisms that determine the correct subcellular localization of proteins targeted to membranes by tail-anchor sequences are poorly defined. Previously, we showed that two isoforms of the tung oil tree [Vernicia (Aleurites) fordii] tail-anchored Cb5 (cytochrome b5) target specifically to ER (endoplasmic reticulum) membranes both in vivo and in vitro [Hwang, Pelitire, Henderson, Andrews, Dyer and Mullen (2004) Plant Cell 16, 3002–3019]. In the present study, we examine the targeting of various tung Cb5 fusion proteins and truncation mutants to purified intracellular membranes in vitro in order to assess the importance of the charged CTS (C-terminal sequence) in targeting to specific membranes. Removal of the CTS from tung Cb5 proteins resulted in efficient binding to both ER and mitochondria. Results from organelle competition, liposome-binding and membrane proteolysis experiments demonstrated that removal of the CTS results in spontaneous insertion of tung Cb5 proteins into lipid bilayers. Our results indicate that the CTSs from plant Cb5 proteins provide ER specificity by preventing spontaneous insertion into incorrect subcellular membranes.
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Research Article|
January 12 2007
The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes
Matthew P. A. Henderson;
Matthew P. A. Henderson
*Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada L8N 3Z5
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Yeen Ting Hwang;
Yeen Ting Hwang
†Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1
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John M. Dyer;
John M. Dyer
‡US Department of Agriculture, Agricultural Research Service, Southern Regional Research Center, New Orleans, LA 70124, U.S.A.
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Robert T. Mullen;
Robert T. Mullen
†Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1
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David W. Andrews
David W. Andrews
1
*Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada L8N 3Z5
1To whom correspondence should be addressed (email Andrewsd@mcmaster.ca).
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Publisher: Portland Press Ltd
Received:
June 28 2006
Revision Received:
September 15 2006
Accepted:
September 19 2006
Accepted Manuscript online:
September 19 2006
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London
2007
Biochem J (2007) 401 (3): 701–709.
Article history
Received:
June 28 2006
Revision Received:
September 15 2006
Accepted:
September 19 2006
Accepted Manuscript online:
September 19 2006
Citation
Matthew P. A. Henderson, Yeen Ting Hwang, John M. Dyer, Robert T. Mullen, David W. Andrews; The C-terminus of cytochrome b5 confers endoplasmic reticulum specificity by preventing spontaneous insertion into membranes. Biochem J 1 February 2007; 401 (3): 701–709. doi: https://doi.org/10.1042/BJ20060990
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