Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.694251
Title: The quality control of transmembrane domains along the secretory pathway
Author: Briant, Kit
ISNI:       0000 0004 5990 4645
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2015
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Abstract:
Protein quality control is crucial to maintaining cellular function. A failure to clear misfolded, aggregation prone proteins can lead to the accumulation of toxic protein aggregates that interfere with cellular pathways and lead to cell death. In addition, the degradation of partially functional proteins can lead to loss of function diseases. Understanding proteins quality control mechanisms is therefore of fundamental importance to understanding these disease pathways. Systems that operate to monitor the structure of soluble protein domains are now relatively well understood. However, in addition to soluble domains, membrane proteins contain regions that span lipid bilayers, and a key question that remains is where and how these transmembrane domains (TMDs) that fail to assemble correctly or are otherwise aberrant are recognised within subcellular compartments. As such, in this study model chimeric proteins containing the luminal and cytoplasmic domain of the single-spanning membrane protein CD8 and exogenous TMDs derived from polytopic membrane proteins were used to investigate the handling of non-native TMDs in the secretory pathway. CD8 chimeras containing non-native TMDs were found to be recognised by endoplasmic reticulum (ER) quality control pathways. Importantly, ER-associated degradation of CD8 chimeras containing exogenous TMDs was reliant upon ubiquitination of cytoplasmic lysine residues prior to retrotranslocation and dislocation from the ER membrane. In contrast, CD8 containing the endogenous TMD but a misfolded luminal domain could be efficiently degraded when cytoplasmic lysines were removed, suggesting that the retrotranslocation mechanisms for these proteins are distinct and defined by the domain which is misfolded. A proportion of the CD8 chimeras containing non-native TMDs were able to exit the ER, and were retrieved to the ER from the Golgi. Golgi-to-ER retrieval was found to be at least partially mediated by Rer1. CD8 chimeras that escaped ER retrieval could also be retained in the Golgi and subsequently degraded in lysosomes, indicating the presence of an as yet undefined TMD-based Golgi quality control checkpoint in mammalian cells. Furthermore, in contrast to WT CD8 which was stable at the plasma membrane, CD8 chimeras containing non-native TMDs that trafficked to the cell surface were rapidly internalised and sorted to lysosomes. This process was largely independent of the cytoplasmic domain of CD8, suggesting signals within the TMD induced internalisation of these CD8 chimeras. The proportion of the CD8 chimeras that trafficked to the plasma membrane, and the stability of the protein at the cell surface, was dependent upon the presence of polar residues within the TMDs, indicating that exposed polar residues in non-native TMDs may alter the handling of proteins at the Golgi and cell surface. Together, these results further our understanding of the mechanisms by which proteins containing aberrant transmembrane domains are handled at multiple subcellular compartments.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.694251  DOI: Not available
Keywords: Cell Biology ; Protein quality control ; transmembrane domain ; ERAD ; endocytosis ; protein trafficking
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