Targeting and expression of mammalian and plasmodium sarco/endoplasmic recticulum calcium pumps
Targeting and expression of mammalian and plasmodium sarco/endoplasmic recticulum calcium pumps
The expression and targeting of a number of sarco/endoplasmic reticulum calcium pumps (SERCAs) were examined. In initial studies, the sub-cellular location of SERCA1 in cultured myotubes from chick embryos was examined. SERCA1 was located in striations that correlated, as expected, with the location of the sarcoplasmic reticulum. To determine whether this compartment also corresponded to the endoplasmic reticulum in these cells the localisation of the transmembrane ER marker calnexin was also examined. Calnexin distribution was clearly localised in a compartment separate from that of SR, being confined to striations running along the length of the myotubules. This compares with the SR striations that are perpendicular to the myotubule axis.
The above studies indicate that ER and SR membranes are distinct within muscle. To determine whether there is any evidence for the continuity of the lumena of ER and SR a constant was produced which targeted enhanced green fluorescent protein (eGFP) to the ER and maintained it there with KDEL, an ER retrieval motif. The eGFP was clearly located in the SR lumena providing evidence for the existence of continuity in the lumena of these two membrane systems.
To examine the targeting of SERCAs from their site of synthesis in ER to their final location in SR the location of a non-muscle isoform SERCA, SERCA3 was examined when transfected into chick myotubes. In order to detect SERCA3 in situ it was necessary to introduce an epitope from SERCA1 into SERCA3 for which an antibody (monoclonal antibody Y/1F4) was available. The tagged sequence of SERCA was expressed in chick myotubes, but despite the fact that this was a non-muscle isoform it still located to the SR as revealed by laser confocal immunofluorescence studies.
University of Southampton
van Goethem, Iris Desirée Aglaia
67755dca-d094-4e74-b5a2-f2d974fe2380
2002
van Goethem, Iris Desirée Aglaia
67755dca-d094-4e74-b5a2-f2d974fe2380
van Goethem, Iris Desirée Aglaia
(2002)
Targeting and expression of mammalian and plasmodium sarco/endoplasmic recticulum calcium pumps.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
The expression and targeting of a number of sarco/endoplasmic reticulum calcium pumps (SERCAs) were examined. In initial studies, the sub-cellular location of SERCA1 in cultured myotubes from chick embryos was examined. SERCA1 was located in striations that correlated, as expected, with the location of the sarcoplasmic reticulum. To determine whether this compartment also corresponded to the endoplasmic reticulum in these cells the localisation of the transmembrane ER marker calnexin was also examined. Calnexin distribution was clearly localised in a compartment separate from that of SR, being confined to striations running along the length of the myotubules. This compares with the SR striations that are perpendicular to the myotubule axis.
The above studies indicate that ER and SR membranes are distinct within muscle. To determine whether there is any evidence for the continuity of the lumena of ER and SR a constant was produced which targeted enhanced green fluorescent protein (eGFP) to the ER and maintained it there with KDEL, an ER retrieval motif. The eGFP was clearly located in the SR lumena providing evidence for the existence of continuity in the lumena of these two membrane systems.
To examine the targeting of SERCAs from their site of synthesis in ER to their final location in SR the location of a non-muscle isoform SERCA, SERCA3 was examined when transfected into chick myotubes. In order to detect SERCA3 in situ it was necessary to introduce an epitope from SERCA1 into SERCA3 for which an antibody (monoclonal antibody Y/1F4) was available. The tagged sequence of SERCA was expressed in chick myotubes, but despite the fact that this was a non-muscle isoform it still located to the SR as revealed by laser confocal immunofluorescence studies.
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Published date: 2002
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Local EPrints ID: 465036
URI: http://eprints.soton.ac.uk/id/eprint/465036
PURE UUID: 511dd3e3-a442-4aee-a4d7-e78f9a8caee4
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Date deposited: 05 Jul 2022 00:18
Last modified: 16 Mar 2024 19:54
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Author:
Iris Desirée Aglaia van Goethem
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