THE PROPEPTIDE OF PROHORMONE CONVERTASE PC2 ACTS AS A TRANSFERABLE AGGREGATION AND MEMBRANE-ASSOCIATION SIGNAL

Prohormone convertase 2 (PC2) is a subtilisin-like protease involved i n the intracellular processing of prohormones and proneuropeptides. Li ke its substrates, it is synthesised as a prepropeptide which undergoe s proteolysis during transit through thr regulated secretory pathway. Previous studies have shown that aggregation and membrane association of proPC2 occurs in a calcium-dependent and pH-dependent manner and th at the pro-region of PC2 may be involved in this process. These events may be involved in the sorting of proteins to the regulated secretory pathway. To investigate this further, we made a chimeric protein cont aining both the signal peptide and pro-region of PC2 and the N-termina l part of alpha 1-antitrypsin, called pro2 alpha 1. PC2, alpha 1-antit rypsin and pro2 alpha 1 were compared with regard to their membrane as sociation and aggregation properties using, respectively, sucrose grad ient centrifugation after expression in Xenopus oocytes, and an in vit ro aggregation assay. The chimeric protein, pro2 alpha 1, underwent lo w-pH-dependent aggregation and membrane association similar to wild-ty pe PC2, Membrane association occurred at pH 5.5 in the absence of calc ium and at pH 6.0 in the presence of 10 mM calcium but not at pH 6.5 o r 7.0. alpha 1-antitrypsin, as expected of a constitutively secreted p rotein, did not aggregate at low pH, nor associate with membranes. Pro 2 alpha 1 thus exhibits the membrane association and aggregation prope rties of PC2, confirming the role of the pro-region in these processes . A series of deletions were performed within the 84-residue propeptid e in order to define the sequences involved. Deletion of amino acids 5 2-77 reduced aggregation but large deletions in the pro-region had onl y a minimal effect on membrane association. These data suggest that se veral regions within the propeptide are important in these events.