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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 B (B). The cells, pulse labeled as described in Materials and  Methods, were chased for up to 8.5 h.  During different 90-min chase intervals, either stimulated (+) or unstimulated (−) secretion was collected (left),  and the cells were then lysed. Equal  fractions of media and cells were immunoprecipitated using anti-ProB, and  one-tenth of these volumes were taken  for precipitation with antiinsulin. The  positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), ProB, and  mature cathepsin B (B) are shown.  Scanning densitometry of these gels  led to the numerical data described in  the text for this representative experiment (of three).
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Figure 1: Stimulus-dependent release from INS cells of peptides immunoprecipitable with antiinsulin (A) or anti– cathepsin B (B). The cells, pulse labeled as described in Materials and Methods, were chased for up to 8.5 h. During different 90-min chase intervals, either stimulated (+) or unstimulated (−) secretion was collected (left), and the cells were then lysed. Equal fractions of media and cells were immunoprecipitated using anti-ProB, and one-tenth of these volumes were taken for precipitation with antiinsulin. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), ProB, and mature cathepsin B (B) are shown. Scanning densitometry of these gels led to the numerical data described in the text for this representative experiment (of three).

Mentions: The trafficking of ProB, a high-affinity ligand for MPRs, was examined in the well-differentiated β-cell line, INS-1 (Asfari et al., 1992), which exhibits regulated exocytosis of insulin in a nearly linear fashion during prolonged (4 h) stimulation (Neerman-Arbez and Halban, 1993). After a 30-min pulse labeling with 35S–amino acids, stimulated or unstimulated secretions from labeled INS cells were sampled at different 90-min chase intervals during an 8.5-h time course. At all chase times, secretagogue-induced exocytosis of newly synthesized insulin (or proinsulin) was obtained (Fig. 1 A, left). During the interval from 15–105 min of chase, stimulus-dependent secretion of ProB could be elicited (Fig. 1 B, left). By quantitative scanning densitometry, the stimulus-dependent release of ProB represented 17% of all immunoprecipitable B obtained at the 105-min chase time and 37.3% of immunoprecipitable ProB obtained at this chase time, while stimulus-dependent secretion of immunoprecipitable insulin–containing peptides was 32% during the same interval. These data indicate that a significant fraction of ProB entered the regulated secretory pathway. Of course, ProB delivery to lysosomes was also ongoing during this chase period, as judged by processing in the cell lysates to the mature 31-kD cathepsin B. Indeed, at times >3 h of chase, labeled ProB was essentially absent from INS cell lysates (Fig. 1 B, right, and data not shown), indicating that its arrival in lysosomes was complete. Consequently, in response to secretagogue addition after 3 h of chase, despite continued regulated exocytosis of labeled insulin (Fig. 1 A, left), INS cells did not release any form of cathepsin B into the medium (Fig. 1 B, left), indicating that neither ProB nor mature cathepsin B remained to any significant extent in the secretory pathway.


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 B (B). The cells, pulse labeled as described in Materials and  Methods, were chased for up to 8.5 h.  During different 90-min chase intervals, either stimulated (+) or unstimulated (−) secretion was collected (left),  and the cells were then lysed. Equal  fractions of media and cells were immunoprecipitated using anti-ProB, and  one-tenth of these volumes were taken  for precipitation with antiinsulin. The  positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), ProB, and  mature cathepsin B (B) are shown.  Scanning densitometry of these gels  led to the numerical data described in  the text for this representative experiment (of three).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Stimulus-dependent release from INS cells of peptides immunoprecipitable with antiinsulin (A) or anti– cathepsin B (B). The cells, pulse labeled as described in Materials and Methods, were chased for up to 8.5 h. During different 90-min chase intervals, either stimulated (+) or unstimulated (−) secretion was collected (left), and the cells were then lysed. Equal fractions of media and cells were immunoprecipitated using anti-ProB, and one-tenth of these volumes were taken for precipitation with antiinsulin. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), ProB, and mature cathepsin B (B) are shown. Scanning densitometry of these gels led to the numerical data described in the text for this representative experiment (of three).
Mentions: The trafficking of ProB, a high-affinity ligand for MPRs, was examined in the well-differentiated β-cell line, INS-1 (Asfari et al., 1992), which exhibits regulated exocytosis of insulin in a nearly linear fashion during prolonged (4 h) stimulation (Neerman-Arbez and Halban, 1993). After a 30-min pulse labeling with 35S–amino acids, stimulated or unstimulated secretions from labeled INS cells were sampled at different 90-min chase intervals during an 8.5-h time course. At all chase times, secretagogue-induced exocytosis of newly synthesized insulin (or proinsulin) was obtained (Fig. 1 A, left). During the interval from 15–105 min of chase, stimulus-dependent secretion of ProB could be elicited (Fig. 1 B, left). By quantitative scanning densitometry, the stimulus-dependent release of ProB represented 17% of all immunoprecipitable B obtained at the 105-min chase time and 37.3% of immunoprecipitable ProB obtained at this chase time, while stimulus-dependent secretion of immunoprecipitable insulin–containing peptides was 32% during the same interval. These data indicate that a significant fraction of ProB entered the regulated secretory pathway. Of course, ProB delivery to lysosomes was also ongoing during this chase period, as judged by processing in the cell lysates to the mature 31-kD cathepsin B. Indeed, at times >3 h of chase, labeled ProB was essentially absent from INS cell lysates (Fig. 1 B, right, and data not shown), indicating that its arrival in lysosomes was complete. Consequently, in response to secretagogue addition after 3 h of chase, despite continued regulated exocytosis of labeled insulin (Fig. 1 A, left), INS cells did not release any form of cathepsin B into the medium (Fig. 1 B, left), indicating that neither ProB nor mature cathepsin B remained to any significant extent in the secretory pathway.

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