Milieu-induced, selective aggregation of regulated secretory proteins in the trans-Golgi network

To study the aggregation of the granins in the secretary path-way, we used the rat pheochromocytoma cell line PC12 . These cells express high levels of CgB and SgII, sulfate these proteins in the TGN, and package them as dense-cored aggregates very efficiently into secretory granules (Lee and Huttner, 1983 ; Rosa et al ., 1985; Rosa et al ., 1989; Tooze and Huttner, 1990 ) . The following experimental approach, referred to as the "standard procedure, was used to study the aggregation of the granins in the TGN. First, short (5 min) pulse labeling of cells with [35S]sulfate was used to label proteins selectively in the TGN (Baeuerle and Huttner, 1987 ; Tooze and Huttner, 1990) , and TGN-derived vesicles were isolated from the pulse-labeled cells by differential and velocity sucrose gradient centrifugation . Second, the [35 S]sulfate-labeled TGN vesicles were permeabilized with saponin in various buffers to be able to expose the TGN lumen to a defined milieu . One buffer had a pH of 7.4 and contained no added calcium ions and is, based on the results described below, referred to as the "nonaggregative milieu ." The other buffer had a pH of 6.4 and contained 10 mM calcium ions, conditions believed to exist in the lumen of the TGN in vivo (Stoeckel et al., 1975 ; Ravazzola, 1976; Mata et al., 1987 ; Anderson and Orci, 1988 ; Roos, 1988 ; see Discussion for "free" vs . "bound" calcium) . This buffer is, based on the results obtained, referred to as the "tiaggregative milieu ." The release of the [35 S]sulfate-labeled granins from the permeabilized TGN vesicles, as revealed by their appearance in the supernatant upon centrifugation, reflects the absence, or reversal, of an aggregated state . Conversely, we interpret the The Journal of Cell Biology, Volume 115, 1991 Figure 1 . Release of granins from the TGN of PC12 cells after permeabilization of the membrane with saponin in nonaggregative milieu . [35 S]sulfate-labeled TGN vesicles were obtained from PC12 cells according to the standard procedure, incubated in nonaggregative milieu in the absence (-) or presence (+) of 0.5 mg/ml saponin, centrifuged, and pellets (P) and supernatants (S) were analyzed by SDS-PAGE followed by protein staining (right) and fluorography (left) . Dots and triangles indicate ER resident proteins released from vesicles contaminating the TGN vesicle preparation .