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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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A significant fraction of ProL in INS cells becomes entrapped in long-term storage in the regulated secretory pathway.  The stimulated (+) and unstimulated (−) secretions from pulselabeled INS cells were collected either during the first 4 h after labeling or after a chase of 20 h to first allow all newly synthesized  ProL molecules to reach their final targets. At the conclusion of  each set, both media and cell lysates were analyzed by immunoprecipitation with anti–cathepsin L. Two exposures of the media,  differing fivefold, are shown in the upper two panels. Note the  disappearance of labeled forms of mature L at later chase times,  and the persistent stimulus-dependent secretion of the precursor.  The positions of ProL and bands comprising mature cathepsin L  (large bracket) are shown.
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Figure 4: A significant fraction of ProL in INS cells becomes entrapped in long-term storage in the regulated secretory pathway. The stimulated (+) and unstimulated (−) secretions from pulselabeled INS cells were collected either during the first 4 h after labeling or after a chase of 20 h to first allow all newly synthesized ProL molecules to reach their final targets. At the conclusion of each set, both media and cell lysates were analyzed by immunoprecipitation with anti–cathepsin L. Two exposures of the media, differing fivefold, are shown in the upper two panels. Note the disappearance of labeled forms of mature L at later chase times, and the persistent stimulus-dependent secretion of the precursor. The positions of ProL and bands comprising mature cathepsin L (large bracket) are shown.

Mentions: To characterize the ultimate fate of intracellular cathepsin L, we compared early chase times to those prolonged up to 1 d to allow maximum opportunity for newly synthesized ProL molecules to reach their proper final targets. Unstimulated and stimulated secretions from INS cells were collected from either 0–4 h of chase or 20–24 h of chase. A short exposure of the media (Fig. 4, top) demonstrated a fivefold stimulated release of ProL during the early chase period (Fig. 4, first and third lanes). In the cell lysates, the arrival of labeled cathepsin L in lysosomes was modest, based on the appearance of the processed mature forms (Fig. 4, bottom, first and third lanes). Remarkably, by 24 h of chase, the newly synthesized mature lysosomal cathepsin L had almost entirely turned over, while intracellular ProL was still detected in a pool that appeared longer lived (Fig. 4, bottom, second and fourth lanes). When the INS cells were exposed to secretagogue during the last 4 h of the experiment, the signal was diminished in proportion to the amount of labeled ProL remaining, but an approximately fivefold stimulated release of ProL was still obtained (Fig. 4, middle), and the fraction of the remaining ProL that exhibited stimulus-dependent exocytosis was not diminished from that seen at the earlier chase time. Moreover, the disappearance of mature L forms, with the sustained presence of ProL, suggested that ProL was contained in a compartment from which delivery to lysosomes was not actively ongoing. Taken together, these findings indicated that a considerable fraction of ProL persisted in secretory granules.


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)

A significant fraction of ProL in INS cells becomes entrapped in long-term storage in the regulated secretory pathway.  The stimulated (+) and unstimulated (−) secretions from pulselabeled INS cells were collected either during the first 4 h after labeling or after a chase of 20 h to first allow all newly synthesized  ProL molecules to reach their final targets. At the conclusion of  each set, both media and cell lysates were analyzed by immunoprecipitation with anti–cathepsin L. Two exposures of the media,  differing fivefold, are shown in the upper two panels. Note the  disappearance of labeled forms of mature L at later chase times,  and the persistent stimulus-dependent secretion of the precursor.  The positions of ProL and bands comprising mature cathepsin L  (large bracket) are shown.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139876&req=5

Figure 4: A significant fraction of ProL in INS cells becomes entrapped in long-term storage in the regulated secretory pathway. The stimulated (+) and unstimulated (−) secretions from pulselabeled INS cells were collected either during the first 4 h after labeling or after a chase of 20 h to first allow all newly synthesized ProL molecules to reach their final targets. At the conclusion of each set, both media and cell lysates were analyzed by immunoprecipitation with anti–cathepsin L. Two exposures of the media, differing fivefold, are shown in the upper two panels. Note the disappearance of labeled forms of mature L at later chase times, and the persistent stimulus-dependent secretion of the precursor. The positions of ProL and bands comprising mature cathepsin L (large bracket) are shown.
Mentions: To characterize the ultimate fate of intracellular cathepsin L, we compared early chase times to those prolonged up to 1 d to allow maximum opportunity for newly synthesized ProL molecules to reach their proper final targets. Unstimulated and stimulated secretions from INS cells were collected from either 0–4 h of chase or 20–24 h of chase. A short exposure of the media (Fig. 4, top) demonstrated a fivefold stimulated release of ProL during the early chase period (Fig. 4, first and third lanes). In the cell lysates, the arrival of labeled cathepsin L in lysosomes was modest, based on the appearance of the processed mature forms (Fig. 4, bottom, first and third lanes). Remarkably, by 24 h of chase, the newly synthesized mature lysosomal cathepsin L had almost entirely turned over, while intracellular ProL was still detected in a pool that appeared longer lived (Fig. 4, bottom, second and fourth lanes). When the INS cells were exposed to secretagogue during the last 4 h of the experiment, the signal was diminished in proportion to the amount of labeled ProL remaining, but an approximately fivefold stimulated release of ProL was still obtained (Fig. 4, middle), and the fraction of the remaining ProL that exhibited stimulus-dependent exocytosis was not diminished from that seen at the earlier chase time. Moreover, the disappearance of mature L forms, with the sustained presence of ProL, suggested that ProL was contained in a compartment from which delivery to lysosomes was not actively ongoing. Taken together, these findings indicated that a considerable fraction of ProL persisted in secretory granules.

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