Job, Christy Amelia; (1995) The biogenesis of regulated secretory organelles. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Membrane proteins located in two types of regulated secretory organelle, the synaptic-like microvesicle and the dense core granule, were analysed in the chromaffin-like neuroendocrine PC 12 tissue culture cell line and in transfected CHO-38 fibroblast and H.Ep.2 epithelial cell lines. Firstly, the movement of newly synthesised synaptophysin (a synaptic-like microvesicle membrane protein) was analysed in CHO-38 and PC 12 cells using a series of pulse-chase immunoprecipitation experiments. Synaptophysin was shown to move rapidly through the secretory pathway of CHO-38 ceils, over the cell surface with similar kinetics to the endogenous synaptophysin in PC 12 cells and then recycle in and out of the cell in the same manner as the transferrin receptor. In PC 12 cells at steady state, however, only a fraction of the synaptophysin was found in the early endosome relative to that in CHO 38 cells, indicating that in PC 12 cells newly synthesised synaptophysin is sorted from the early endosome to the synaptic-like microvesicle. Secondly, the distribution of p-selectin (a secretory granule membrane protein) and p- selectin deletion mutants were investigated in PC 12 cells and transfected H.Ep.2 cells using double label immunofluorescence and confocal light microscopy. In PC 12 cells, wild type p-selectin was distributed in granules while a mutant with the terminal fourteen amino acids removed from the cytoplasmic tail was directed initially to the cell surface and then to a perinuclear localisation which may be lysosomal. This implies that these C-terminal amino acid residues partially or completely encode the granule targeting signal. In H.Ep.2 cells both mutant and wild type p-selectins were directed to the lysosome. Finally, the targeting of a p-selectin horseradish peroxidase chimaera was determined by electron microscopy in transfected PC 12 and H.Ep.2 cells and found to be in dense core granules and multivesicular bodies respectively.

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