Purification and biochemical characterization of a protein complex from the sea urchin, Strongylocentrotus purpuratus : possible functional role for the yolk granule organelle during embryonic development

Perera, Aruni Shamalee (2003) Purification and biochemical characterization of a protein complex from the sea urchin, Strongylocentrotus purpuratus : possible functional role for the yolk granule organelle during embryonic development. Masters thesis, Memorial University of Newfoundland.

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Abstract

The yolk granule is the most abundant membrane-bound organelle present in the eggs and embryos of many animals including avians, amphibians, insects, mollusks and echinoderms. The sea urchin yolk granules comprise about one-third of the volume of the egg. The classical view of the yolk granule is that it provides nutrition to the growing embryo, but the finding that the composition of the sea urchin yolk granule does not change until the later stages of larval development changed this view, suggesting that the yolk granules are not just benign storage organelles, but might be involved in some other cellular events occurring during embryonic development. -- Several studies have demonstrated that sea urchin yolk granules harbor several components destined for export. Therefore, we hypothesized that the yolk granules might be involved in transportation and fusion events. We have isolated a protein of 240 kDa present in the yolk granules of eggs from the sea urchin, Strongylocentrotus purpuratus, by ion exchange chromatography using the anion exchange resin, Q-Sepharose Fast Flow. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of this protein under non-reducing conditions revealed that this was a complex composed of three polypeptides of 160 kDa, 120 kDa and 90 kDa. Western blots performed using the anti-toposome antibody demonstrated that the 240 kDa protein complex was the toposome which was proteolytically processed, while the 160 kDa polypeptide was the major yolk granule protein derived from it. Peptide mapping confirmed that the 240 kDa toposome was the precursor for the 160 kDa major yolk granule protein. -- We have biochemically characterized the calcium-dependent phospholipid binding and vesicular aggregating activity of the 240 kDa protein complex. As revealed by liposome binding assays, the 240 kDa protein complex bound phospholipids in a calcium-dependent manner. Liposome aggregation assays demonstrated that this 240 kDa protein complex was capable of driving vesicular aggregation, which was also found to be a calcium-dependent process. The yolk granule aggregation assays allowed us to demonstrate that this aggregating activity was a physiologically relevant process. The anti-toposome antibody could specifically inhibit the calcium dependent phospholipid binding, liposome aggregation and the yolk granule aggregating activity of the 240 kDa protein complex. The exposure of yolk granules to trypsin inhibited aggregation suggesting that this process was driven by protein present in the outer surface of the yolk granule membranes. Analysis of the yolk granule membranes isolated by density gradient ultracentrifugation suggested that the membranes were rich in high molecular weight polypeptides of 160 k, 130 k, 120 k and 90 k. Collectively, these data suggest that the 240 kDa protein or the polypeptides derived from it could be the factor/s involved in vesicular aggregation and transportation events involving the yolk granule.

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