Analysis of the sorting of secretory proteins to the regulated secretory pathway. A subcellular fractionation approach.

Abstract

AbstractProtein secretion is a fundamental process common to all animal cells, whereby a subset of newly synthesized proteins is constitutively secreted into the external environment. In specialized cell types, such as those found in endocrine and neuronal tissues, protein secretion also occurs in a regulated fashion: A unique subset of newly synthesized proteins is stored in secretory granules within the cell and is only secreted after an external stimulus is received by the cell. To study these two pathways of secretion, the constitutive and the regulated, cell lines that are capable of both types of secretion have proved invaluable. We have been studying protein secretion in the neuroendocrine cell line PC12 (rat pheochromocytoma), with the aim of dissecting both the constitutive and regulated pathway at a molecular level. To characterize these two pathways in PC12 cells, the approaches we have adopted employ biosynthetic labeling of newly synthesized proteins (see Subheading 3.2.), treatment with well-characterized drugs (see Subheading 3.3.), and subcellular fractionation techniques (see Subheading 3.5.). To identify the molecules that are involved in the functioning of theses two pathways, we have used a variety of in vitro assays that faithfully reconstitute the cellular events (see Subheadings 3.8.–3.9.).KeywordsPC12 CellSecretory GranuleHomogenization BufferLaemmli Sample BufferSucrose Gradient CentrifugationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.