Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic b -Cells

In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 b -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 b -granules, while immunolabeling for cathepsin L, but not B, persists in mature b -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. L ysosomes are digestive organelles found in all eukaryotic cells (Kornfeld and Mellman, 1989), whereas secretory granules are apparent only in a subgroup of cell types specialized for regulated secretion of certain proteins that are stored at high concentration (Kelly, 1985; Burgess and Kelly, 1987). In recent years, studies of specialized cell types have reported structural and functional overlap between lysosomes and secretory granules (Taugner and Hackenthal, 1988; Bonifacino et al., 1989; Burkhardt et al., 1989, 1990), including the idea that a fraction of lysosomal enzymes/proenzymes may exist in the regulated secretory pathway of endocrine and exocrine tissues under physiological conditions (Brands et al., 1982; Docherty et al., 1984; Tooze et al., 1991; Hirano et al., 1992). To date, the trafficking of lysosomal prohydrolases has been well studied only in cells that produce few or no secretory granules. From these studies, the basic schema of posttranslational carbohydrate modification and recognition by mannose-6-phosphate receptors (MPRs) 1 is now generally accepted (von Figura and Hasilik, 1986; von Figura, 1991). By contrast, two substantially different kinds of hypotheses, which are not mutually exclusive, have prevailed concerning the sorting of regulated secretory proteins. The “sorting for entry” theories include the possibility of one or more conserved receptors whose primary function would be to bind lumenal content proteins in the TGN and “usher” them (Kelly, 1987) or “zipper” them (Kelly, 1985; Kelly, 1991; Tooze and Stinchcombe, 1992) into forming granules. Thus, it has been envisaged in sorting for entry models that at the time of entry into immature granules (IGs), regulated secretory proteins are either bound to receptors (Moore et al., 1989) or bound to other regulated secretory proteins that are bound to receptors (Palmer and Christie, 1992; Pimplikar and Huttner, 1992; Leblond et al., 1993; Yoo, 1993; Natori and Huttner, 1996; Cool et al., 1997), while nongranule secretory proteins reAddress all correspondence to Peter Arvan, Diabetes Research Center and Division of Endocrinology, Albert Einstein College of Medicine, Bronx, NY 10461. Tel.: (718) 430-8685. Fax: (718) 430-8557. 1. Abbreviations used in this paper : CDand CI-MPR, cation-dependent and -independent mannose-6-phosphate receptors; IG, immature granules; M6P, mannose-6-phosphate; MPR, mannose-6-phosphate receptor; ProB and L, procathepsin B and L. on July 7, 2017 jcb.rress.org D ow nladed fom