Integral membrane proteins play a wide range of functions which are essential in many cellular processes. The biogenesis of membrane proteins occurs mainly at the endoplasmic reticulum (ER) in eukaryotes and is monitored by quality control (QC) systems that are able to distinguish between native and non-native conformation. Misfolded or misassembled proteins are identified by QC systems at the ER and also at later points in the secretory pathway, and then targeted for degradation. Membrane proteins may possess lumenal, cytosolic and transmembrane domains (TMD), and it is proposed that distinct QC mechanisms exist for monitoring folding in these different locations. To date, most studies have focused on the QC of misfolded domains in the ER lumen. Very little is known about the mechanisms that monitor folding / assembly of TMD and identify abnormal TMDs for degradation. The aim of this work was to establish a model protein to examine TMD QC at the ER. A chimeric protein, CD8TM4-ha, based on CD8alpha with the endogenous TMD replaced with a single TMD (TM4) of a multispanning membrane protein was generated.This chimera had a steady state ER localisation and was rapidly degraded. Comparison with control chimeras showed that this was due to signals within the TMD, suggesting that TM4 in the context of CD8TM4-ha exposes a "misfolding/misassembly‟ signal. Biochemical and microscopy experiments showed that not all of the CD8TM4-ha was statically retained in the ER, but at least some of the protein cycled through the Golgi, where it received a post-translational modification, most likely O-glycosylation. The CD8TM4-ha lacking this modification was degraded via a proteasome dependent pathway (likely to be ERAD), whilst the Golgi modified forms were degraded at least partly by lysosomes. These results suggest that QC systems at the ER and post-ER compartments are able to recognise abnormal TMDs and target them for degradation. A small siRNA screen identified several candidate proteins that contribute to CD8TM4-ha QC. Amongst these were the ERAD E3 ubiquitin ligase HRD1 and its accessory factor SEL1-L, whose depletion inhibited degradation of CD8TM4-ha. This suggests that the HRD1 complex plays a key role in ERAD of proteins with defective TMDs. On the other hand, knocking down the lectin chaperone calnexin lead to the accumulation of CD8TM4-ha in post-ER compartments, suggesting that it may contribute to recognition and retention of aberrant TMDs in the ER. Future studies will be aimed at defining the relationship between these components, and defining in more detail the QC pathways that recognise CD8TM4-ha at the ER and beyond.
Date of Award | 1 Aug 2011 |
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Original language | English |
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Awarding Institution | - The University of Manchester
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Supervisor | Eileithyia Swanton (Supervisor) |
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Quality control of transmembrane domains in the secretory pathway
Otsuka, Y. (Author). 1 Aug 2011
Student thesis: Master of Philosophy