The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic β cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6–phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by ∼90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in β cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595–608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261–1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR–ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.

In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 β-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature β-granules, while immunolabeling for cathepsin L, but not B, persists in mature β-granules. By contrast, in islets from normal male SpragueDawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulusdependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.

Abstract. We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C-peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Aryan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. Z BioL Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (tv2 -1.5 h) cornpared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20~ temperature block reveals a tl~ ~< 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway. W E have been studying the existence of a unique pathway of protein secretion that exists in cells that also exhibit exocytotic discharge of secretory granules. This route is clearly distinct from true constitutive secretion in that it conveys content proteins that have been sequestered within the lumen of immature granules, and exhibits the same polarized targeting as the regulated secretory pathway (Arvan and Castle, 1987;Aryan and Lee, 1991). Nevertheless, this route is detected most readily under unstimulated conditions. As demonstrated by electron microscope autoradiography, this pathway seems to be related to the presence of early granule forms (immature secretory granules [IGs]~; Saipeter and Farquhar, 1981) rather than mature granules (Aryan and Chang, 1987). We have termed this novel route the constitutive-like pathway, in an attempt to distinguish it both from regulated exocytosis as well as true constitutive secretion (Aryan et al., 1991). The constitutive-like pathway appears to account for obser- vations of regulated protein secretion from two parallel intracellular pools (Rothman and Issenman, 1974;Roberge and Beaudoin, 1982), as well as preferential unstimulated release of newly synthesized peptide hormones over those stored in matur...