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

• Author / Creator

  Kraus, Allison

• 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.

• Subjects / Keywords

  • Calnexin
  • Endoplasmic reticulum
  • Myelin

• Graduation date

  Fall 2011

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3XF0Q

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.