Targeting of the sarco/endoplasmic calcium ATPase
Targeting of the sarco/endoplasmic calcium ATPase
Nucleated eukaryotic cells have two types of calcium pump belonging to the family of P-type ATPases; sarco/endoplasmic calcium ATPases (SERCAs) are located in the endo/sarcoplasmic reticulum (ER/SR) whereas plasma membrane calcium ATPases (PMCAs) are located at the plasma membrane. The mechanism(s) by which SERCAs are maintained in the ER/SR is unknown and in this study I have identified the segment of SERCA1b containing the retention/retrieval signal by producing SERCA/PMCA chimeras linked to enhanced green fluorescent protein (eGFP). Pairs of chimeras have been produced in which the structure of one chimera is the obverse of its partner. For example, one chimera is comprised of the central domain of SERCA linked to the N- and C-termini of PMCA. This contrasts with a construct containing N- and C-termini of SERCA in combination with the central domain of PMCA. When comparing the chimeras one of a pair should lose the retention/retrieval signal, while the other should still contain it. In addition, to determine whether SERCA is truly retained in the ER, or retrieved from a post-ER compartment, co-locations studies have been performed using antibodies against ERGIC-53, which is a marker for the ER-Golgi intermediate compartment (ERGIC). Laser confocal microscopy clearly shows that SERCA-eGFP has entered the ERGIC, and this is confirmed by cell fractionation experiments. This strongly suggests that retrieval plays a significant role in maintaining SERCA 1b in the ER.
University of Southampton
Newton, Tom
aa900957-c933-4f82-862d-ac43ece7e156
2003
Newton, Tom
aa900957-c933-4f82-862d-ac43ece7e156
Newton, Tom
(2003)
Targeting of the sarco/endoplasmic calcium ATPase.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Nucleated eukaryotic cells have two types of calcium pump belonging to the family of P-type ATPases; sarco/endoplasmic calcium ATPases (SERCAs) are located in the endo/sarcoplasmic reticulum (ER/SR) whereas plasma membrane calcium ATPases (PMCAs) are located at the plasma membrane. The mechanism(s) by which SERCAs are maintained in the ER/SR is unknown and in this study I have identified the segment of SERCA1b containing the retention/retrieval signal by producing SERCA/PMCA chimeras linked to enhanced green fluorescent protein (eGFP). Pairs of chimeras have been produced in which the structure of one chimera is the obverse of its partner. For example, one chimera is comprised of the central domain of SERCA linked to the N- and C-termini of PMCA. This contrasts with a construct containing N- and C-termini of SERCA in combination with the central domain of PMCA. When comparing the chimeras one of a pair should lose the retention/retrieval signal, while the other should still contain it. In addition, to determine whether SERCA is truly retained in the ER, or retrieved from a post-ER compartment, co-locations studies have been performed using antibodies against ERGIC-53, which is a marker for the ER-Golgi intermediate compartment (ERGIC). Laser confocal microscopy clearly shows that SERCA-eGFP has entered the ERGIC, and this is confirmed by cell fractionation experiments. This strongly suggests that retrieval plays a significant role in maintaining SERCA 1b in the ER.
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Published date: 2003
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Local EPrints ID: 465069
URI: http://eprints.soton.ac.uk/id/eprint/465069
PURE UUID: ef04d79f-4953-4405-b3d2-52475b5ba220
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Date deposited: 05 Jul 2022 00:21
Last modified: 16 Mar 2024 19:55
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Author:
Tom Newton
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