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.
Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic β-Cells
1. Abbreviations used in this paper: CD- and 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.
4. Notably, our data in pancreatic β-cells do not specifically support the function of a non–carbohydrate-dependent lysosomal proenzyme receptor (McIntyre and Erickson, 1991, 1993; McIntyre et al., 1994) in sorting out of the biosynthetic pathway, although whether such a receptor might be involved in trafficking steps that occur within the endocytic pathway remains an open question.
2. The quantitative data from transgenic and control animals were obtained from film scanning of two independent experiments, in which all individual values were within 30% of the mean. Because there is conservation of 14 cysteines and four methionines between ProB and the mature form, no correction factor to account for isotope recovery was applied to our calculations.
The authors are greatly indebted to Drs. P. Halban and M. NeermanArbez for providing and instructing us on the use of INS cells, as well as important suggestions and discussions during the course of this work. We are grateful to Drs. J. Mort, O. Madsen, G. Sahagian, A. Saluja, and F. Brodsky for generously providing the antibodies used in these studies. We thank T. Broers-Vendrig and J. Griffiths for technical assistance, as well as R. Scriwanek and M. Niekerk (Free University, Amsterdam) for preparation of the electron micrographs. We also thank members of the Arvan lab and Professor H.J. Geuze (University of Utrecht, The Netherlands) for advice and support.
Address 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.
3. MPRs are involved in proenzyme delivery to endosomes (Griffiths et al., 1988). If stimulated prohydrolase secretion were to derive from β-cell endosomes, the reduced affinity of ProL for MPRs should have decreased ProL delivery from the biosynthetic pathway to the stimulatable compartment, which in turn would diminish apparent regulated secretion, whereas in fact, just the opposite was observed (Figs. 3 and 5). Further, long-term storage of ProL in endosomes would not be an expected consequence of a low affinity for MPRs (Kornfeld and Mellman, 1989).
Regina Kuliawat, Judith Klumperman, Thomas Ludwig, Peter Arvan; Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic β-Cells. J Cell Biol 5 May 1997; 137 (3): 595–608. doi: https://doi.org/10.1083/jcb.137.3.595
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