Targeting the calcium ATPase to the endoplasmic reticulum

Abstract

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.

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