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Title: Targeting the calcium ATPase to the endoplasmic reticulum
Author: Watson, Helen Rachel
ISNI:       0000 0004 2685 2180
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2009
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The sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps calcium from the cytoplasm into the lumen of the endoplasmic or sarcoplasmic reticulum (ER/SR), removing excess Ca2+ from the cytoplasm and replenishing ER/SR Ca2+ stores. SERCA is located in both the ER and the ER-Golgi intermediate compartment, and so is likely maintained in the ER by retrieval. To locate the ER retrieval signal(s) in SERCA, a series of chimeric calcium pumps have been constructed. Sections of SERCA were replaced with corresponding sequence from its plasma membrane counterpart; plasma membrane calcium ATPase (PMCA). Replacing the C-terminus of SERCA with corresponding PMCA sequence results in mistargeting of the protein to the plasma membrane. The opposite construct (consisting of PMCA with the C-terminus replaced by that of SERCA) is located in the ER, suggesting that the ER retrieval signal lies towards the C-terminus of the protein. Many of the chimeras built were located in the ER. This is likely to be due to protein misfolding in some cases. Attempts were made to detect the unfolded protein response in cells expressing chimeras by measuring levels of the chaperone protein BiP. BiP upregulation was only seen when the unfolded protein response was induced pharmacologically, and not in cells expressing chimeras. More subtle mutagenesis was then carried out to assess the role of the tenth transmembrane domain of SERCA in ER retrieval and CD8 reporter constructs were used to study the tenth transmembrane domains of SERCA and PMCA. The study then focussed on determining the mechanism by which SERCA is retrieved to the ER. Rer1p and BAP31 are both candidate receptors for the retrieval of SERCA. An antibody to two epitopes in human Rer1p was raised and characterised. Immunoprecipitation and cross-linking showed that although Rer1p appears not to interact with SERCA, BAP31 shows a potential interaction and therefore could be involved in the retrieval of the calcium pump to the ER.
Supervisor: East, John ; Lee, Anthony Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QH301 Biology