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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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Stimulus-dependent exocytosis of mature  β-granules releases an increased fraction of ProB from  the islets of transgenic MPRdeficient mice. Islets from  CD-MPR–deficient mice were  pulse labeled and chased  overnight in the absence of  secretagogues to allow the labeled proinsulin and ProB to  chase to their final destinations, before collecting sequential 30-min periods of unstimulated (−) and stimulated (+) secretion. See text for details. Secretion of labeled insulin and immunoprecipitable ProB were  analyzed by SDS-PAGE and fluorography.
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Figure 12: Stimulus-dependent exocytosis of mature β-granules releases an increased fraction of ProB from the islets of transgenic MPRdeficient mice. Islets from CD-MPR–deficient mice were pulse labeled and chased overnight in the absence of secretagogues to allow the labeled proinsulin and ProB to chase to their final destinations, before collecting sequential 30-min periods of unstimulated (−) and stimulated (+) secretion. See text for details. Secretion of labeled insulin and immunoprecipitable ProB were analyzed by SDS-PAGE and fluorography.

Mentions: Therefore, islets from CD-MPR–deficient or control mice were pulse labeled and chased in the absence of secretagogues for a sufficient period to allow the labeled proinsulin and ProB to chase into mature granules and lysosomes, respectively. Upon subsequent analysis of stimulus-dependent secretion, MPR-deficient mice exhibited a clear increase in stimulated discharge of ProB along with insulin from mature β-granules (Fig. 12). In age-matched control mice, ProB was barely detectable in the stimulusdependent secretion from mature β-granules, representing only ∼3% of all immunoprecipitable forms of cathepsin B,2 whereas in the islets from CD-MPR–deficient mice, this value was ∼11%, i.e., increasing the missorted fraction of ProB nearly fourfold.


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)

Stimulus-dependent exocytosis of mature  β-granules releases an increased fraction of ProB from  the islets of transgenic MPRdeficient mice. Islets from  CD-MPR–deficient mice were  pulse labeled and chased  overnight in the absence of  secretagogues to allow the labeled proinsulin and ProB to  chase to their final destinations, before collecting sequential 30-min periods of unstimulated (−) and stimulated (+) secretion. See text for details. Secretion of labeled insulin and immunoprecipitable ProB were  analyzed by SDS-PAGE and fluorography.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139876&req=5

Figure 12: Stimulus-dependent exocytosis of mature β-granules releases an increased fraction of ProB from the islets of transgenic MPRdeficient mice. Islets from CD-MPR–deficient mice were pulse labeled and chased overnight in the absence of secretagogues to allow the labeled proinsulin and ProB to chase to their final destinations, before collecting sequential 30-min periods of unstimulated (−) and stimulated (+) secretion. See text for details. Secretion of labeled insulin and immunoprecipitable ProB were analyzed by SDS-PAGE and fluorography.
Mentions: Therefore, islets from CD-MPR–deficient or control mice were pulse labeled and chased in the absence of secretagogues for a sufficient period to allow the labeled proinsulin and ProB to chase into mature granules and lysosomes, respectively. Upon subsequent analysis of stimulus-dependent secretion, MPR-deficient mice exhibited a clear increase in stimulated discharge of ProB along with insulin from mature β-granules (Fig. 12). In age-matched control mice, ProB was barely detectable in the stimulusdependent secretion from mature β-granules, representing only ∼3% of all immunoprecipitable forms of cathepsin B,2 whereas in the islets from CD-MPR–deficient mice, this value was ∼11%, i.e., increasing the missorted fraction of ProB nearly fourfold.

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