Velocity and isopycnic gradient centrifugation were employed to fractionate post-nuclear supernatants rapidly prepared from PC12 cells in order to characterize areas of the endoplasmic reticulum involved in various aspects of intracellular Ca2+ homeostasis. The endoplasmic reticulum Ca2+ pumping activity, defined by three properties studied in parallel in the isolated fractions; thapsigargin-sensitive uptake of 45Ca2+, Ca2+- dependent, thapsigargin-sensitive protein phosphorylation and Western blotting of sarcoplasmic reticulum calcium ATPase (SERCA) 2b and putative SERCA3 ATPases, was concentrated primarily in a few fractions located at the top and toward the bottom of velocity and isopycnic gradients, respectively. The endoplasmic reticulum Ca2+ release channel, the inositol 1,4,5- trisphosphate receptor, was concentrated in the same fractions as the Ca2+ pumps, and additionally in a few fractions distinctly poor in SERCAs. In contrast, two lumenal markers (protein disulfide isomerase and calreticulin, the major Ca2+ storage protein of non-muscle endoplasmic reticulum) were enriched in the middle fractions of the velocity gradients while calnexin, a Ca2+-binding membrane protein, was more widely distributed throughout the gradients. These results document a considerable degree of functional and compositional heterogeneity in the endoplasmic reticulum of neurosecretory PC12 cells. Even in the limited areas that appear specialized for rapid Ca2+ uptake and release the ratio between pumps and channels varies considerably. Within the rest of the system, insulated from short-term fluctuations of Ca2+ concentration, Ca2+-binding proteins appear to be extensively distributed, in agreement with the idea that the Ca2+ content of the endoplasmic reticulum serves multiple functions.
ASJC Scopus subject areas