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A structural and functional investigation of calnexin and its unique cytoplasmic domain Open Access

Descriptions

Other title
Subject/Keyword
endoplasmic reticulum
calnexin
myelin
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Kraus, Allison
Supervisor and department
Michalak, Marek (Biochemistry)
Examining committee member and department
Bleackley, Chris (Biochemistry)
Young, Howard (Biochemistry)
Hobman, Tom (Cell Biology)
Argon, Yair (UPenn, Pathology and Laboratory Medicine)
Department
Department of Biochemistry
Specialization

Date accepted
2011-08-26T17:39:48Z
Graduation date
2011-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
Calnexin is a ubiquitously expressed endoplasmic reticulum chaperone that in conjunction with the similar endoplasmic reticulum chaperone calreticulin and protein disulfide isomerase ERp57, is responsible for protein folding and quality control in the secretory pathway. We generated a calnexin-deficient mouse model to use a loss-of function approach to study the role of calnexin. Calnexin-deficient mice are 30-50% smaller than their wild-type littermates and demonstrate neurological abnormalities characterized by gait disturbance, ataxia and a rolling walk. Neuron number, growth and function were unimpaired in the absence of calnexin. However, electron micrograph analysis indicates decompacted, disorganized myelin sheaths. Nerve conduction velocities were correspondingly reduced in sensory and motor neurons. The role of calnexin in myelination is a major discovery that provides a novel candidate gene for myelin disease and the resulting pathologies. Our work highlights a previously unidentified substrate specificity of a ubiquitous chaperone, showcasing the importance of specific chaperones and negating the notion that quality control is a redundant process. Investigation of the role calnexin plays in myelin formation and maintenance will help us understand myelin biology and the aberrant processes that result in dysmyelination and disease. Calnexin is composed of distinct functional and structural domains including an N-terminal globular and extended arm P-domain (N+P domain) that forms the protein folding module, a transmembrane domain, and a long C-terminal cytoplasmic tail. The N+P domain of calnexin are reminiscent of another quality control chaperone, calreticulin, and calnexin and calreticulin are known to share folding substrates. However, the transmembrane domain and cytoplasmic tail of calnexin are unique and as calnexin plays a non-redundant role in myelin and myelin protein quality control, the C-tail could confer calnexin’s specificity for membrane myelin proteins. To study the function of the C-tail, we employed techniques to look at the structure and biophysical characteristics as well as protein-protein interactions of the calnexin C-tail. A yeast-2-hybrid screen with the C-tail as bait identified UBC9, a SUMOylation E2 ligase, as a protein that interacts with the C-tail. Further biochemical studies reveal that the C-tail interacts with components of the SUMOylation machinery including UBC9, and can be SUMOylated in vivo. SUMOylation is a novel post-translational modification of the C-tail. Understanding the role of calnexin and its unique cytoplasmic tail will provide mechanistic insight into the function of a critical quality control chaperone, including understanding the specific role it plays in myelination.
Language
English
DOI
doi:10.7939/R3XF0Q
Rights
License granted by Allison Kraus (kraus@ualberta.ca) on 2011-08-24T18:31:25Z (GMT): Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of the above terms. The author reserves all other publication and other rights in association with the copyright in the thesis, and except as herein provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
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