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Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta-cells.

Kuliawat R, Klumperman J, Ludwig T, Arvan P - J. Cell Biol. (1997)

Bottom Line: By contrast, in islets from normal male Sprague-Dawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulus-dependent 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.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Center and Division of Endocrinology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 beta-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 beta-granules, while immunolabeling for cathepsin L, but not B, persists in mature beta-granules. By contrast, in islets from normal male Sprague-Dawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulus-dependent 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.

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Unstimulated and stimulated secretion of newly synthesized insulin (A), ProB (B), and ProL (C) from pancreatic  islets of normal male Sprague-Dawley rats. The islets, untreated  or pretreated with tunicamycin, were pulse labeled for 30 min.  During 30–90 min of chase, the islets were exposed either to unstimulated (−) or stimulated (+) conditions. Secretion of proinsulin + insulin was analyzed by immunoprecipitation as described (Arvan et al., 1991). Immunoprecipitable ProB and ProL  were analyzed by SDS-PAGE and fluorography. After tunicamycin treatment, ProB was secreted as the unglycosylated form.
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Figure 13: Unstimulated and stimulated secretion of newly synthesized insulin (A), ProB (B), and ProL (C) from pancreatic islets of normal male Sprague-Dawley rats. The islets, untreated or pretreated with tunicamycin, were pulse labeled for 30 min. During 30–90 min of chase, the islets were exposed either to unstimulated (−) or stimulated (+) conditions. Secretion of proinsulin + insulin was analyzed by immunoprecipitation as described (Arvan et al., 1991). Immunoprecipitable ProB and ProL were analyzed by SDS-PAGE and fluorography. After tunicamycin treatment, ProB was secreted as the unglycosylated form.

Mentions: Regulated secretory tissues and cell lines do have a bona fide TGN compartment (Tooze and Huttner, 1990) through which newly synthesized proteins travel before their arrival in post-Golgi compartments (Kuliawat and Arvan, 1992; Miller et al., 1992; Rosa et al., 1992; Xu and Shields, 1993; Huang and Arvan, 1994). Moreover, the potential for clathrin-coated vesicle budding is shared by both the TGN and IGs (Fig. 9). These facts raise an important question (considered further in the Discussion): Specifically, does the sorting of lysosomal hydrolase precursors from TGN to endosomes always have the same efficiency in regulated secretory cells generally, and in β-cells in particular? With this question in mind, we have been interested to explore the extent of prohydrolase entry into β-cell IGs from islets prepared from different animal models. We therefore proceeded to examine the islets of normal male Sprague-Dawley rats. Surprisingly, in spite of the brisk stimulated release of labeled proinsulin and insulin (Fig. 13 A), even at early chase times the presence of newly synthesized ProB in IGs (as detected by stimulus-dependent secretion; Fig. 13 B, left) was obviously diminished from that observed in INS cells (Fig. 1) or the islets of normal mice (Kuliawat and Arvan, 1994). Similar “negative” results were obtained upon examination of a second high-affinity MPR ligand, the ∼75-kD β-glucuronidase precursor (data not shown). By contrast, after tunicamycin treatment such that newly synthesized ProB was unglycosylated, ProB entrance into the regulated secretory pathway resumed (Fig. 13 B, right). Moreover, in the case of ProL, even without tunicamycin treatment, stimulus-dependent secretion could be detected in the islets of normal male Sprague Dawley rats (Fig. 13 C). These data indicate that although lysosomal proenzymes may enter IGs in abundance, this entrance can vary considerably. Thus, IGs may functionally extend the TGN but do not serve as a mandatory intermediate in the trafficking of hydrolase precursors to lysosomes in all cases in regulated secretory cells.


Differential sorting of lysosomal enzymes out of the regulated secretory pathway in pancreatic beta-cells.

Kuliawat R, Klumperman J, Ludwig T, Arvan P - J. Cell Biol. (1997)

Unstimulated and stimulated secretion of newly synthesized insulin (A), ProB (B), and ProL (C) from pancreatic  islets of normal male Sprague-Dawley rats. The islets, untreated  or pretreated with tunicamycin, were pulse labeled for 30 min.  During 30–90 min of chase, the islets were exposed either to unstimulated (−) or stimulated (+) conditions. Secretion of proinsulin + insulin was analyzed by immunoprecipitation as described (Arvan et al., 1991). Immunoprecipitable ProB and ProL  were analyzed by SDS-PAGE and fluorography. After tunicamycin treatment, ProB was secreted as the unglycosylated form.
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Related In: Results  -  Collection

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Figure 13: Unstimulated and stimulated secretion of newly synthesized insulin (A), ProB (B), and ProL (C) from pancreatic islets of normal male Sprague-Dawley rats. The islets, untreated or pretreated with tunicamycin, were pulse labeled for 30 min. During 30–90 min of chase, the islets were exposed either to unstimulated (−) or stimulated (+) conditions. Secretion of proinsulin + insulin was analyzed by immunoprecipitation as described (Arvan et al., 1991). Immunoprecipitable ProB and ProL were analyzed by SDS-PAGE and fluorography. After tunicamycin treatment, ProB was secreted as the unglycosylated form.
Mentions: Regulated secretory tissues and cell lines do have a bona fide TGN compartment (Tooze and Huttner, 1990) through which newly synthesized proteins travel before their arrival in post-Golgi compartments (Kuliawat and Arvan, 1992; Miller et al., 1992; Rosa et al., 1992; Xu and Shields, 1993; Huang and Arvan, 1994). Moreover, the potential for clathrin-coated vesicle budding is shared by both the TGN and IGs (Fig. 9). These facts raise an important question (considered further in the Discussion): Specifically, does the sorting of lysosomal hydrolase precursors from TGN to endosomes always have the same efficiency in regulated secretory cells generally, and in β-cells in particular? With this question in mind, we have been interested to explore the extent of prohydrolase entry into β-cell IGs from islets prepared from different animal models. We therefore proceeded to examine the islets of normal male Sprague-Dawley rats. Surprisingly, in spite of the brisk stimulated release of labeled proinsulin and insulin (Fig. 13 A), even at early chase times the presence of newly synthesized ProB in IGs (as detected by stimulus-dependent secretion; Fig. 13 B, left) was obviously diminished from that observed in INS cells (Fig. 1) or the islets of normal mice (Kuliawat and Arvan, 1994). Similar “negative” results were obtained upon examination of a second high-affinity MPR ligand, the ∼75-kD β-glucuronidase precursor (data not shown). By contrast, after tunicamycin treatment such that newly synthesized ProB was unglycosylated, ProB entrance into the regulated secretory pathway resumed (Fig. 13 B, right). Moreover, in the case of ProL, even without tunicamycin treatment, stimulus-dependent secretion could be detected in the islets of normal male Sprague Dawley rats (Fig. 13 C). These data indicate that although lysosomal proenzymes may enter IGs in abundance, this entrance can vary considerably. Thus, IGs may functionally extend the TGN but do not serve as a mandatory intermediate in the trafficking of hydrolase precursors to lysosomes in all cases in regulated secretory cells.

Bottom Line: By contrast, in islets from normal male Sprague-Dawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulus-dependent 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.

View Article: PubMed Central - PubMed

Affiliation: Diabetes Research Center and Division of Endocrinology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 beta-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 beta-granules, while immunolabeling for cathepsin L, but not B, persists in mature beta-granules. By contrast, in islets from normal male Sprague-Dawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulus-dependent 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.

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