CHARACTERIZATION OF THE GOLGI-COMPLEX CLEARED OF PROTEINS IN TRANSIT AND EXAMINATION OF CALCIUM-UPTAKE ACTIVITIES

To characterize endogenous molecules and activities of the Golgi compl ex, proteins in transit were > 99% cleared from rat hepatocytes by usi ng cycloheximide (CHX) treatment. The loss of proteins in transit resu lted in condensation of the Golgi cisternae and stacks. Isolation of a stacked Golgi fraction is equally efficient with or without proteins in transit [control (CTL SGF1) and cycloheximide (CHX SGF1)]. Electron microscopy and morphometric analysis showed that > 90% of the element s could be positively identified as Golgi stacks or cisternae. Biochem ical analysis showed that the cis-, medial-, trans-, and TGN Golgi mar kers were enriched over the postnuclear supernatant 200- to 400-fold w ith and 400- to 700-fold without proteins in transit. To provide infor mation on a mechanism for import of calcium required at the later stag es of the secretory pathway, calcium uptake into CTL SGF1 and CHX SGF1 was examined. All calcium uptake into CTL SGF1 was dependent on a tha psigargin-resistant pump not resident to the Golgi complex and a thaps igargin-sensitive pump resident to the Golgi. Experiments using CHX SG F1 showed that the thapsigargin-resistant activity was a plasma membra ne calcium ATPase isoform in transit to the plasma membrane and the th apsigargin-sensitive pump was a sarcoplasmic/endoplasmic reticulum cal cium ATPase isoform. In vivo both of these calcium ATPases function to maintain millimolar levels of calcium within the Golgi lumen.