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Volume 271, Number 46, Issue of November 15, 1996 pp. 29304-29311
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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The Endoplasmic Reticulum in PC12 Cells
EVIDENCE FOR A MOSAIC OF DOMAINS DIFFERENTLY SPECIALIZED IN Ca²⁺ HANDLING

(Received for publication, April 2, 1996, and in revised form, September 4, 1996)

From the Department of Pharmacology and B. Ceccarelli Centre, University of Milan, the CNR Centre of Molecular and Cellular Pharmacology, and DIBIT, San Raffaele Scientific Institute, via Olgettina 58, 20132 Milano, Italy

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.

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