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Title: Ca²⁺-dependent interactions in synaptic vesicle endocytosis
Author: Falconer, Helen Louise
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2008
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Endocytosis proteins contain a number of different specialised domains which control protein-protein interactions at specific steps. A variety of these domains were used in a proteomic screen using GST-pull down assays from nerve terminal lysates to investigate whether Ca2+ controlled any specific interactions. Many interactions appeared to occur only in the presence of Ca2+, however this was attributed to non-specific binding due to the presence of ZnSO4­. Two new interactions were identified that bind SH3 domains only in the absence of Ca2+, the adaptor protein caskin I and a protein known as “similar to KIAA0856”. Since SH3 domain containing proteins had Ca2+-dependent interactions, these were examined in more detail. Most proteins bound to SH3 domains only in the absence of Ca2+. These proteins included caskin I, synaptojanin, amphiphysin I, dynamin I and synapsin I. However an unidentified 90 kDa band bound only in the presence of Ca2+ suggesting Ca2+ positively and negatively regulates interactions. These interactions were characterised further by examining the amount of Ca2+ required to stimulate or inhibit the above associations. Ca2+ binding of endophilin II has been proposed to control its protein-protein interactions. A number of complementary techniques were used to examine the Ca2+ affinity of SH3 domains including endophilin. These techniques included 45Ca2+ overlay assays, tyrosine and tryptophan fluorescence and equilibrium dialysis. From all of the fusion proteins investigated the SH3 domains appeared to bind 45Ca2+ however the full length proteins did not. Tyrosine and tryptophan fluorescence showed a Ca2+ dependent fluorescence shift suggesting a conformational change on the addition of Ca2+, meaning that Ca2+ may bind to the SH3 domains. Finally equilibrium dialysis showed that no endophilin GST fusion protein bound to Ca2+. From these results it is still open to question whether endophilin binds Ca2+.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available