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

If aggregation of regulated secretary proteins is a sorting event, constitutively released proteins should be excluded from the aggregates. If, in the case of the granins, a rise in calcium and a decrease in pH are the parameters that trigger their aggregation in thelumen ofthe TGN ofPC12 cells, constitutively secreted proteins present at the same time in the TGN should not undergo aggregation in the aggregative milieu . The only constitutive marker in PC12 cells which is sulfated and thus can be selectively labeled in the TGN is the hsPG (Tooze and Huttner, 1990) . Unfortunately, the hsPG was found to be membrane associated in the TGN since it was not solubilized when TGN vesicles obtained from PC12 cells pulse labeled for 5 min with [11S]sulfate were extracted with carbonate at pH 11.0 ( Fig. 4 A, left) . We therefore employed 4-methylumbelliferyl R-D-xyloside (xyloside) sulfatelabeled TGN vesicles were obtained from xyloside-treated PC12 cells according to the standard procedure, subjected to carbonate extraction at pH 11, centrifuged, and the pellets (P) and supernatants (S) were analyzed by SDS-PAGE using 7.5% (-xyloside) or 15% (+ xyloside) gels, followed by fluorography. The asterisks indicate a sulfated =120-kD membrane protein distinct from CgB which is largely masked by the hsPG in the absence of xyloside and which has an electrophoretic mobility similar to CgB on 15% gels . to induce the synthesis of free sulfated GAG chains as a bulk flow marker. Xyloside competes with the core proteins of proteoglycans as an acceptor for GAG chains (Lohmander and Hascall, 1979) , and xyloside-induced GAG chains have been found to be secreted largely via the constitutive pathway in AtT20 cells (Burgess and Kelly, 1984) . Three lines of evidence showed that xyloside-induced GAG chains are soluble in the TGN and can serve as a constitutively secreted bulk flow marker in PC12 cells . First, carbonate extraction at pH 11.0 of TGN vesicles, prepared from PC12 cells pretreated with 1 mM xyloside for 2 h and pulse labeled for 5 min with [a 5 S]sulfate, showed that the sulfated GAG chains, which had an M, between 5,000 and 20,000, were soluble ( Fig. 4 A, right) . Second, pulse-chase experiments with PC12 cells showed that the release of the sulfated GAG chains into the medium followed constitutive kinetics. When xyloside-treated PO2 cells pulse labeled for 5 min with [35 S]sulfate were chased at 37°C for 30 and 60 min, -63 and 88%, respectively, of the total [35S]sulfate-labeled GAG chains were released into the medium (Fig . 4 B) . The secretion of the GAG chains was inhibited at 20°C, as is the case for the hsPG ('Iboze and Hutmer, 1990) . When PC12 cells were pulse labeled at 20°C for 5 min with ["S]sulfate and chased at 20°C, the secretion of the labeled GAG chains was completely inhibited over a 60min chase period (Fig . 4 B) . When the temperature was then raised to 37°C, -78% of the total [ 35 S]sulfate-labeled GAG chains were released into the medium during the following 30 min of chase . Xyloside treatment of PC12 cells did not affect the efficient sorting of the granins in these cells since neither CgB nor SgII were secreted during the chase at 37°C (data not shown) . Third, as shown in Fig. 4 (Tooze and Huttner, 1990) . This was consistent with the observation that the sulfation of GAG chains is a turns-Golgi-specific event (Kimura et al ., 1984) .