pH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules.
We have previously characterized the processing of secretogranin II (SgII) in PC12 cells that were stably transfected with the endopeptidase PC2. Here we show that processing of SgII can be observed in isolated immature secretory granules (ISGs) derived from this cell line in a temperature- and ATP-dependent manner. The stimulatory effect of ATP on processing can be attributed to the activation of the vacuolar H(+)-ATPase and a concomitant decrease in intragranular pH. The immature secretory granule therefore provides an adequate environment for correct processing of SgII by PC2. The rate of SgII processing was strongly dependent on the intragranular pH, suggesting that processing of SgII can be used as a pH indicator for the granule interior. A standard curve was prepared using SgII processing in ISGs equilibrated at a range of pH values. The extent of processing in ISGs incubated in the presence of ATP at physiological pH was compared with the standard curve, and the intragranular pH was determined. From these observations, we propose an intragranular pH of 6.3 +/- 0.1 for ISGs in a physiological buffer in the presence of ATP. Hence, the pH of ISGs seems to be similar to the pH of the trans-Golgi network (TGN) and is clearly higher than the pH of mature secretory granules (pH 5.0-5.5). Interestingly, no processing of SgII could be observed in a membrane fraction that is highly enriched in TGN under conditions for which processing was readily obtained in isolated ISGs.
Urbé
S
S
Imperial Cancer Research Fund, London, U.K.
Dittié
A S
AS
Tooze
S A
SA
D000818:Animals; D002864:Chromogranins; D003594:Cytoplasmic Granules / Q000201:enzymology*; D006056:Golgi Apparatus / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009363:Neoplasm Proteins / Q000378:metabolism*; D009479:Neuropeptides / Q000378:metabolism*; D016716:PC12 Cells; D045706:Proprotein Convertase 2; D011506:Proteins / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000378:metabolism; D051381:Rats; D013381:Subtilisins / Q000378:metabolism*
D002864:Chromogranins; D009363:Neoplasm Proteins; D009479:Neuropeptides; D011506:Proteins; D013381:Subtilisins; D045706:Proprotein Convertase 2; D006180:Proton-Translocating ATPases