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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 release from INS cells of peptides  immunoprecipitable with antiinsulin (A) or anti–cathepsin L (B).  The cells were pulse labeled and chased as in Fig. 1, except that  the stimulated (+) or unstimulated (−) medium collections began  at 5 min of chase, were 60 min in duration, and were terminated  at 2 h of chase. Only the medium is shown. While stimulated secretion of ProL is seen at all chase times, note the progression of  processing from proinsulin to insulin in the regulated secretory  pathway. Measurement of stimulus-dependent secretion during a  1-h period for L-containing peptides (∼16%) was in a similar  range to that of insulin-containing peptides (∼21%) in this experiment. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), ProL, and bands  comprising mature cathepsin L (Mr ∼32,000 and ∼27,000, respectively, large bracket) are shown.
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Figure 2: Stimulus-dependent release from INS cells of peptides immunoprecipitable with antiinsulin (A) or anti–cathepsin L (B). The cells were pulse labeled and chased as in Fig. 1, except that the stimulated (+) or unstimulated (−) medium collections began at 5 min of chase, were 60 min in duration, and were terminated at 2 h of chase. Only the medium is shown. While stimulated secretion of ProL is seen at all chase times, note the progression of processing from proinsulin to insulin in the regulated secretory pathway. Measurement of stimulus-dependent secretion during a 1-h period for L-containing peptides (∼16%) was in a similar range to that of insulin-containing peptides (∼21%) in this experiment. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), ProL, and bands comprising mature cathepsin L (Mr ∼32,000 and ∼27,000, respectively, large bracket) are shown.

Mentions: We next used INS cells to examine the trafficking of ProL, a low-affinity ligand for MPRs (Dong and Sahagian, 1990; Lazzarino and Gabel, 1990). Because of the possibility of rapid intracellular transport of ProL (Dong et al., 1989; Kane, 1993) and long stimulation times required to elicit granule exocytosis from INS cells (Neerman-Arbez and Halban, 1993), we initially screened for ProL secretion beginning 5 min after a 30-min pulse labeling (Fig. 2). As shown during the first 2 h of chase, while proinsulin to insulin conversion was clearly ongoing (Fig. 2 A), secretagogue-induced secretion of ProL was also observed (Fig. 2 B). Evidently, like ProB, newly synthesized ProL could also enter a stimulus-dependent secretory pathway in INS cells. However, unlike ProB, at chase times up to 6 h, only ∼20% of ProL ever reached lysosomes, as judged by conversion in the cell lysates to the bands comprising processed forms of mature L (Fig. 3). A 3-h secretagogue exposure elicited secretion of ⩾50% of granule insulin from INS cells, in agreement with previous reports (NeermanArbez and Halban, 1993). Importantly, the residual fraction of labeled ProL (Fig. 3, right) exhibited comparable stimulated secretion (Fig. 3, left).


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 release from INS cells of peptides  immunoprecipitable with antiinsulin (A) or anti–cathepsin L (B).  The cells were pulse labeled and chased as in Fig. 1, except that  the stimulated (+) or unstimulated (−) medium collections began  at 5 min of chase, were 60 min in duration, and were terminated  at 2 h of chase. Only the medium is shown. While stimulated secretion of ProL is seen at all chase times, note the progression of  processing from proinsulin to insulin in the regulated secretory  pathway. Measurement of stimulus-dependent secretion during a  1-h period for L-containing peptides (∼16%) was in a similar  range to that of insulin-containing peptides (∼21%) in this experiment. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), ProL, and bands  comprising mature cathepsin L (Mr ∼32,000 and ∼27,000, respectively, large bracket) are shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Stimulus-dependent release from INS cells of peptides immunoprecipitable with antiinsulin (A) or anti–cathepsin L (B). The cells were pulse labeled and chased as in Fig. 1, except that the stimulated (+) or unstimulated (−) medium collections began at 5 min of chase, were 60 min in duration, and were terminated at 2 h of chase. Only the medium is shown. While stimulated secretion of ProL is seen at all chase times, note the progression of processing from proinsulin to insulin in the regulated secretory pathway. Measurement of stimulus-dependent secretion during a 1-h period for L-containing peptides (∼16%) was in a similar range to that of insulin-containing peptides (∼21%) in this experiment. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), ProL, and bands comprising mature cathepsin L (Mr ∼32,000 and ∼27,000, respectively, large bracket) are shown.
Mentions: We next used INS cells to examine the trafficking of ProL, a low-affinity ligand for MPRs (Dong and Sahagian, 1990; Lazzarino and Gabel, 1990). Because of the possibility of rapid intracellular transport of ProL (Dong et al., 1989; Kane, 1993) and long stimulation times required to elicit granule exocytosis from INS cells (Neerman-Arbez and Halban, 1993), we initially screened for ProL secretion beginning 5 min after a 30-min pulse labeling (Fig. 2). As shown during the first 2 h of chase, while proinsulin to insulin conversion was clearly ongoing (Fig. 2 A), secretagogue-induced secretion of ProL was also observed (Fig. 2 B). Evidently, like ProB, newly synthesized ProL could also enter a stimulus-dependent secretory pathway in INS cells. However, unlike ProB, at chase times up to 6 h, only ∼20% of ProL ever reached lysosomes, as judged by conversion in the cell lysates to the bands comprising processed forms of mature L (Fig. 3). A 3-h secretagogue exposure elicited secretion of ⩾50% of granule insulin from INS cells, in agreement with previous reports (NeermanArbez and Halban, 1993). Importantly, the residual fraction of labeled ProL (Fig. 3, right) exhibited comparable stimulated secretion (Fig. 3, left).

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