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

Morphological work has established that the aggregation of regulated secretory proteins typically occurs in the TGN Table II . Effect ofAggregative Milieu on SgII in the TGN ofControl and Hormone-treated GH4C1 Cells GH4C1 cells cultured for 2 d in the absence (control) or presence (hormonetreated) of'estrodiol, insulin, and EGF were labeled for 4 min with [3 'S]sulfair andthen processed according to the standard procedure except that fractions 3+4 of thevelocity sucrose gradient were used. TGN vesicles were incubated with Triton X-100 in nonaggregative or aggregative milieu, centrifuged, and pellets and supernatants were analyzed by SDS-PAGE followed by fluorography. SgII Only the region of the fluorogram containing the SgIl band is shown. Bar, 10 Am . (Farquhar and Palade, 1981; Orci et al., 1987; Tooze et al., 1987) . In the present study, we have developed a system in which biochemical parameters involved in this aggregation process could be investigated . By using [31S]sulfate to label the granins selectively in the TGN, and by permeabilizing TGN vesicles prepared from such cells with saponin, we could specify the milieu in the lumen of the TGN and thus identify parameters of the milieu that are involved in the aggregation process. We found that a decrease in pH and an increase in the concentration of calcium ions, two parameters that we believe are relevant for granin aggregation in the TGN in vivo, prevented the release of the granins from saponin-permeabilized TGN vesicles. The lack of release was not due to low pH and high calcium interfering with the membrane permeabilization since other lumenal proteins and GAG chains were released under these conditions. Further evidence for the permeabilization ofthe TGN membrane comes from the observation (not shown) that in the absence of leupeptin there was substantial and sometimes complete proteolysis ofthe granins, which most likely was caused by lysosomal proteases that had access to the granins after saponin permeabilization of membranes. Several lines of evidence indicate that the lack ofsolubility of the granins after permeabilization of the TGN membrane in the low pH-, high calcium milieu reflected the conservation of the granin aggregates known to exist in this compartment (Tooze and Huttner, 1990) . First, no pelleting of the granins was observed when these proteins were first allowed to diffuse from permeabilized TGN vesicles in nonaggregative milieu and then exposed to aggregative milieu, or when a partially purified preparation of these proteins was incubated in aggregative milieu . These observations make it unlikely that after permeabilization of the TGN vesicles in aggregative milieu, the granins diffused from the lumen of the TGN and then formed aggregates which sedimented independently of the TGN vesicles. Second, EM on immature secretory granules indicated that membrane permeabilization in aggregative milieu allowed the recovery of densecored structures which appeared similar to the bona fide dense cores of the starting material. Consistent with this observation, when the membrane of mature secretory granules was permeabilized in aggregative milieu, the granins remained in an aggregated state although these proteins are readily solubilized after exocytosis which releases them into a neutral pH-, low calcium milieu .