Targeting the calcium ATPase to the endoplasmic reticulum

The sarco/endoplasmic reticulum calcium ATPase (SERCA) pumps calcium from the<br/>cytoplasm into the lumen of the endoplasmic or sarcoplasmic reticulum (ER/SR), removing<br/>excess Ca2+ from the cytoplasm and replenishing ER/SR Ca2+ stores. SERCA is located in<br/>both the ER and the ER-Golgi intermediate compartment, and so is likely maintained in the<br/>ER by retrieval. To locate the ER retrieval signal(s) in SERCA, a series of chimeric calcium<br/>pumps have been constructed. Sections of SERCA were replaced with corresponding sequence<br/>from its plasma membrane counterpart; plasma membrane calcium ATPase (PMCA).<br/>Replacing the C-terminus of SERCA with corresponding PMCA sequence results in<br/>mistargeting of the protein to the plasma membrane. The opposite construct (consisting of<br/>PMCA with the C-terminus replaced by that of SERCA) is located in the ER, suggesting that<br/>the ER retrieval signal lies towards the C-terminus of the protein. Many of the chimeras built<br/>were located in the ER. This is likely to be due to protein misfolding in some cases. Attempts<br/>were made to detect the unfolded protein response in cells expressing chimeras by measuring<br/>levels of the chaperone protein BiP. BiP upregulation was only seen when the unfolded<br/>protein response was induced pharmacologically, and not in cells expressing chimeras. More<br/>subtle mutagenesis was then carried out to assess the role of the tenth transmembrane domain<br/>of SERCA in ER retrieval and CD8 reporter constructs were used to study the tenth<br/>transmembrane domains of SERCA and PMCA. The study then focussed on determining the<br/>mechanism by which SERCA is retrieved to the ER. Rer1p and BAP31 are both candidate<br/>receptors for the retrieval of SERCA. An antibody to two epitopes in human Rer1p was raised<br/>and characterised. Immunoprecipitation and cross-linking showed that although Rer1p appears<br/>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.

Name dc:type.qualificationname

Ph.D.

Level dc:type.qualificationlevel

doctoral

Grantor dc:publisher.institution

University of Southampton

Year dc:date.issued

2009

Author dc:creator

• Watson, Helen Rachel

Advisors dc:contributor.advisor

• East, J.M.
• Lee, A.G.