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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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Ultrathin cryosection of the Golgi (G) region of a mouse islet β-cell, triple immunolabeled for proinsulin (5-nm gold), cathepsin B (10-nm gold), and insulin (15-nm gold). Insulin label was found over all compartments of the secretory pathway. Granule profiles  with the presence of proinsulin label were used to define IGs (I), while those with insulin labeling only and a variable-sized halo between the content and surrounding membrane were considered mature granules (M). A lysosome (L) is seen to be labeled exclusively  for cathepsin B, although additional cathepsin B label can be seen in IGs. mito, mitochondrion. Bar, 200 nm.
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Figure 7: Ultrathin cryosection of the Golgi (G) region of a mouse islet β-cell, triple immunolabeled for proinsulin (5-nm gold), cathepsin B (10-nm gold), and insulin (15-nm gold). Insulin label was found over all compartments of the secretory pathway. Granule profiles with the presence of proinsulin label were used to define IGs (I), while those with insulin labeling only and a variable-sized halo between the content and surrounding membrane were considered mature granules (M). A lysosome (L) is seen to be labeled exclusively for cathepsin B, although additional cathepsin B label can be seen in IGs. mito, mitochondrion. Bar, 200 nm.

Mentions: In studying the distribution of immunoreactive cathepsins in pancreatic β-cells, one theoretical concern was the ability to discriminate between insulin-containing lysosomes (Orci et al., 1984b; Halban et al., 1987) and true secretory granules. In fact, lysosomes, tubular lysosomes, and crinophagic lysosomes were all unmistakably distinguishable from secretory granules by immunoelectron microscopy, as well as by conventional morphological criteria (Fig. 6, A–C). Further, to reliably differentiate IGs from mature granules, we capitalized on the existence of a monoclonal antibody to proinsulin, whose immunoreactivity is lost upon processing to insulin (Orci et al., 1986). Thus, when examining β-cells of mouse islets, mature granules that have little residual proinsulin remain unlabeled, while IGs of different ages contain varying degrees of labeling (Fig. 7). When double or triple labeling with this mAb was performed along with anti–cathepsin B, cathepsin immunoreactivity was clearly evident in early granules (Figs. 7 and 8, A and B). In addition, proinsulin-positive IGs not infrequently exhibited electron-dense coated membrane buds similar to coated vesicles forming in the TGN. Using an antibody to clathrin, coated buds on both IGs and the TGN were specifically decorated (Fig. 9). However, not only did such buds appear absent from mature granules, but cathepsin B immunolabeling also appeared far less abundant over mature granules (Figs. 7 and 8, A and B).


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)

Ultrathin cryosection of the Golgi (G) region of a mouse islet β-cell, triple immunolabeled for proinsulin (5-nm gold), cathepsin B (10-nm gold), and insulin (15-nm gold). Insulin label was found over all compartments of the secretory pathway. Granule profiles  with the presence of proinsulin label were used to define IGs (I), while those with insulin labeling only and a variable-sized halo between the content and surrounding membrane were considered mature granules (M). A lysosome (L) is seen to be labeled exclusively  for cathepsin B, although additional cathepsin B label can be seen in IGs. mito, mitochondrion. Bar, 200 nm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139876&req=5

Figure 7: Ultrathin cryosection of the Golgi (G) region of a mouse islet β-cell, triple immunolabeled for proinsulin (5-nm gold), cathepsin B (10-nm gold), and insulin (15-nm gold). Insulin label was found over all compartments of the secretory pathway. Granule profiles with the presence of proinsulin label were used to define IGs (I), while those with insulin labeling only and a variable-sized halo between the content and surrounding membrane were considered mature granules (M). A lysosome (L) is seen to be labeled exclusively for cathepsin B, although additional cathepsin B label can be seen in IGs. mito, mitochondrion. Bar, 200 nm.
Mentions: In studying the distribution of immunoreactive cathepsins in pancreatic β-cells, one theoretical concern was the ability to discriminate between insulin-containing lysosomes (Orci et al., 1984b; Halban et al., 1987) and true secretory granules. In fact, lysosomes, tubular lysosomes, and crinophagic lysosomes were all unmistakably distinguishable from secretory granules by immunoelectron microscopy, as well as by conventional morphological criteria (Fig. 6, A–C). Further, to reliably differentiate IGs from mature granules, we capitalized on the existence of a monoclonal antibody to proinsulin, whose immunoreactivity is lost upon processing to insulin (Orci et al., 1986). Thus, when examining β-cells of mouse islets, mature granules that have little residual proinsulin remain unlabeled, while IGs of different ages contain varying degrees of labeling (Fig. 7). When double or triple labeling with this mAb was performed along with anti–cathepsin B, cathepsin immunoreactivity was clearly evident in early granules (Figs. 7 and 8, A and B). In addition, proinsulin-positive IGs not infrequently exhibited electron-dense coated membrane buds similar to coated vesicles forming in the TGN. Using an antibody to clathrin, coated buds on both IGs and the TGN were specifically decorated (Fig. 9). However, not only did such buds appear absent from mature granules, but cathepsin B immunolabeling also appeared far less abundant over mature granules (Figs. 7 and 8, A and B).

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.

Show MeSH
Related in: MedlinePlus