Identification and characterization of CLC-1 interacting proteins
| Autor: | Hao-Han Wu, 吳皓涵 |
|---|---|
| Rok vydání: | 2013 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 CLC-1 is a voltage-gated chloride channel widely expressed in skeletal muscles. The opening probability (Po) of CLC-1 increases with membrane depolarization. The physiological role of CLC-1 is to stabilize membrane potential and to induce membrane repolarization. Previous studies have shown mutations in the gene encoding CLC-1 lead to myotonia. Currently, the detailed mechanism regarding protein synthesis, protein folding, and membrane trafficking of CLC-1 remain unclear. The goal of this thesis is to understand the biosynthesis of CLC-1 channel by searching for novel channel-binding proteins. We employed yeast-two hybrid (Y2H) screening of a mouse skeletal muscle cDNA library. Candidate proteins identified from Y2H screening were subjected to further verification with GST pull-down and co-immunoprecipitation assays. Two candidate proteins were confirmed: P#37 and F#8. F#8 is known be involved in endoplasmic reticulum (ER) protein quality control. Our immunofluorescence image analyses also showed that F#8 and CLC-1 indicated that the CBS2 (cystathionine β-synthase 2) domain in the C-terminus of CLC-1 may be essential for the interaction with F#8. Upon co-expression of CLC-1 and F#8 in HEK293T cells, the expression level of CLC-1 was significantly increased, indicate that F#8 may be involved in the ER quality control of CLC-1. There are still a lot of candidate proteins which need to be further characterized in order to understand the biochemical pathways of CLC-1 in cells. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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