Proinsulin endoproteolysis confers enhanced targeting of processed insulinto the regulated secretory pathway

Recently, two different prohormone-processing enzymes, prohormone convertas e 1 (PC1) and carboxypeytidase E, have been implicated in enhancing the sto rage of peptide hormones in endocrine secretory granules. It is important t o know the extent to which such molecules may act as "sorting receptors" to allow the selective trafficking of cargo proteins from the trans-Golgi net work into forming granules, versus acting as enzymes that may indirectly fa cilitate intraluminal storage of processed hormones within maturing granule s. GH4C1 cells primarily store prolactin in granules; they lack PC1 and are defective for intragranular storage of transfected proinsulin. However, pr oinsulin readily enters the immature granules of these cells. Interestingly , GH4C1 clones that stably express modest levels of PC1 store more proinsul in-derived protein in granules. Even in the presence of PC1, a sizable port ion of the proinsulin that enters granules goes unprocessed, and this porti on largely escapes granule storage. Indeed, all of the increased granule st orage can be accounted for by the modest portion converted to insulin. Thes e results are not unique to GH4C1 cells; similar results are obtained upon PC1 expression in PC12 cells as well as in AtT20 cells (in which PC1 is exp ressed endogenously at higher levels). An in vitro assay of protein solubil ity indicates a difference in the biophysical behavior of proinsulin and in sulin in the PC1 transfectants. We conclude that processing to insulin, fac ilitated by the catalytic activities of granule proteolytic enzymes, assist s in the targeting (storage) of the hormone.