Targeting of the (ca2+-mg2+)-atpase to the sarco-endoplasmic retculum

Black, John Peter James (1999) Targeting of the (ca2+-mg2+)-atpase to the sarco-endoplasmic retculum. University of Southampton, Doctoral Thesis.

Abstract

The mechanism by which ER Ca²⁺ pumps are retained is unknown. In this thesis, two approaches have been used to identify this mechanism.

SERCA1b/PMCA3 chimeras were constructed and expressed in COS-7 cells. The chimeras were designed in pairs so that each chimera, in which SERCA structures had been replaced by PMCA domains, was matched with a corresponding PMCA structure in which the same domains were replaced by SERCA domains. Functionality of the chimeras was confirmed by calcium-dependent phosphorylation and uptake of calcium into microsomes to show that the pumps were correctly folded. This was important since mis-folded proteins are retained in the ER. All constructs were found to be active indicating correct folding.

Localisation studies in COS-7 cells showed that for two of the three chimeric pairs (chimeras I/II and chimeras IV/V), two were targeted to the ER (chimeras I and V) and the other two to the plasma membrane (chimeras II and IV). The third chimeric pair (chimeras III/VI), failed to show a clear difference in location. These results suggest that residues located within the first 212 residues of its sequence mediate ER retention of SERCA1. The chimeras were then expressed in MDCKs in which the plasma membrane of the cells were labelled. This failed to elucidate any further information.

The first 212 residues of SERCA1 contain the first two putative transmembrane helices M1 and M2. A motif found in transmembrane helix M1 of SERCA1 has been suggested as a possible targeting signal. This motif was mutated, but there was no evidence to suggest a role in ER retention.

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