Involvement of the membrane lipid bilayer in sorting prohormone convertase2 into the regulated secretory pathway

Prohormone convertase 2 (PC2) is a neuroendocrine-specific protease involve d in the intracellular maturation of prohormones and proneuropeptides. PC2 is synthesised as a proprotein (proPC2) that undergoes proteolysis, aggrega tion and membrane association during its transit through the regulated secr etory pathway. We have previously shown that the pro region of proPC2 plays a key role in its aggregation and membrane association. To investigate thi s further, we determined the binding properties of a peptide containing ami no acids 45-84 of proPC2 (proPC2(45-84)) to trans-Golgi network/granule-enr iched membranes from the AtT20 cell line, Removal of peripheral membrane pr oteins or hydrolysis of integral membrane proteins did not affect the bindi ng properties of proPC2(45-84). Rather, proPC2(45-84) was shown to bind to protein-free liposomes in a pH- and Ca2+- dependent manner. To identify the component of the lipid bilayer involved in this membrane association, we u sed chromaffin-granule membranes and studied the binding properties of the endogenous PC2. Treatment of the membranes with saponin, a cholesterol-depl eting detergent, failed to extract PC2 from the membranes, whereas chromogr anin A (CgA) was removed. Treatment of the membranes with Triton X-100 yiel ded a low-density detergent-insoluble fraction enriched in PC2, but not CgA . The detergent-insoluble fraction also contained glycoprotein III, known t o be part of the lipid rafts (membrane microdomains rich in sphingolipids). Finally, sphingolipid depletion of AtT20 cells resulted in the mis-sorting of PC2, suggestive of a link between the association of PC2 with lipid raf ts in the membrane and its sorting into the regulated secretory pathway.