In vitro condensation-sorting of enzyme proteins isolated from rat pancreatic acinar cells

To study the process of granule formation in pancreatic acinar cells in more detail we have established an in vitro system in which the whole complement of enzyme proteins released from isolated zymogen granules is mixed with a tracer amount of the same biosynthetically labeled proteins and is incubated at conditions prevailing in either pre-Golgi (pH 7.5) or trans-Golgi (pH 5.9) compartments. Condensation of the proteins into dense cores is assayed and quantitated after centrifugation of the mixture at 13000g and separation of the proteins in both the supernatant and the pellet by 2D-gel electrophoresis. At pH 7.5 about 1% of the total protein-bound radioactivity can be sedimented into the pellet and this increases 5-fold at pH 5.9 with similar sedimentation efficiency for individual enzyme proteins. The usual assumption that all aggregated proteins can be sedimented and thus only the pellet is representative for pH-dependent condensation has to be modified by the fine structural analysis of both the supernatant and pellet fraction at pH 7.5 and 5.9. Small particulate complexes form already in the supernatant at pH 7.5 which are not sedimented to a large extent into the pellet. At pH 5.9 aggregates of a homogeneous size of about 0.6 to 0.8 microm formed in the supernatant while the pellet is composed of sheets and vesicles of membranes studded with dense core particles of about 20 to 30nm size. The pH-dependent protein condensation is a stepwise process starting with the formation of small dense core particles already at pH 8.0/7.5 which then progressively aggregate to form larger cores at pH 6.0/5.0. These aggregates can only be sedimented employing higher centrifugal forces. In the condensation process of pancreatic enzyme proteins calcium ions exert an effect only at pH 7.5, leading to somewhat larger dense particles, while potassium ions are inhibitory both in protein condensation and in the binding of particles to membranes. The process of pH-dependent protein condensation is reversible and can be performed repetitively. The sedimentation of condensed proteins can be increased by the addition of isolated zymogen granule membranes. Thus the in vitro system allows the analysis of two related processes in granule formation: the condensation of secretory proteins into granule cores and their binding to the granule membrane.