Limits...
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.

Show MeSH
Stimulus-dependent release  from INS cells treated with tunicamycin.  Untreated INS cells (left) or those pretreated with tunicamycin (right) were  pulse labeled and chased for 3.5 h before  60-min collections of stimulated (+) or unstimulated (−) secretion, or the resulting  cell lysates were analyzed by immunoprecipitation with antiinsulin (A) or anti– cathepsin L (B). For INS cells treated with  tunicamycin, samples were intentionally  double loaded to enhance the sensitivity of  detection of mature cathepsin L forms.  Note that after tunicamycin treatment, the  amount of intracellular precursor increased disproportionately, as did the  amount of stimulus-dependent secretion  of ProL. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), glycosylated  and unglycosylated ProL, and bands comprising glycosylated and unglycosylated mature cathepsin L (large brackets) are  shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139876&req=5

Figure 5: Stimulus-dependent release from INS cells treated with tunicamycin. Untreated INS cells (left) or those pretreated with tunicamycin (right) were pulse labeled and chased for 3.5 h before 60-min collections of stimulated (+) or unstimulated (−) secretion, or the resulting cell lysates were analyzed by immunoprecipitation with antiinsulin (A) or anti– cathepsin L (B). For INS cells treated with tunicamycin, samples were intentionally double loaded to enhance the sensitivity of detection of mature cathepsin L forms. Note that after tunicamycin treatment, the amount of intracellular precursor increased disproportionately, as did the amount of stimulus-dependent secretion of ProL. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), glycosylated and unglycosylated ProL, and bands comprising glycosylated and unglycosylated mature cathepsin L (large brackets) are shown.

Mentions: In the experiment shown in Fig. 5, INS cells were pulse labeled and chased for 3.5 h before a 1-h period of stimulus-dependent secretion was analyzed. The left panels show the results from control cells that are similar to Fig. 3, insofar as partial delivery to lysosomes was detected. The right panels were performed in parallel from INS cells treated with tunicamycin. In this case, samples were intentionally double loaded to enhance the sensitivity of detection of mature cathepsin L forms. Nevertheless, it was clear that the arrival of unglycosylated L in lysosomes was profoundly inhibited, while there was a commensurate increase in the amount of intracellular ProL. Evidently, in these cells, despite the low affinity of ProL for MPRs, that portion of ProL which is successfully targeted to lysosomes still uses a carbohydrate-dependent sorting mechanism. Moreover, the increase in missorted ProL after tunicamycin treatment resulted in a proportionate increase in the pool of unglycosylated precursor in the regulated secretory pathway, as demonstrated by markedly increased stimulus-dependent secretion (Fig. 5, right).


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 treated with tunicamycin.  Untreated INS cells (left) or those pretreated with tunicamycin (right) were  pulse labeled and chased for 3.5 h before  60-min collections of stimulated (+) or unstimulated (−) secretion, or the resulting  cell lysates were analyzed by immunoprecipitation with antiinsulin (A) or anti– cathepsin L (B). For INS cells treated with  tunicamycin, samples were intentionally  double loaded to enhance the sensitivity of  detection of mature cathepsin L forms.  Note that after tunicamycin treatment, the  amount of intracellular precursor increased disproportionately, as did the  amount of stimulus-dependent secretion  of ProL. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), glycosylated  and unglycosylated ProL, and bands comprising glycosylated and unglycosylated mature cathepsin L (large brackets) are  shown.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Stimulus-dependent release from INS cells treated with tunicamycin. Untreated INS cells (left) or those pretreated with tunicamycin (right) were pulse labeled and chased for 3.5 h before 60-min collections of stimulated (+) or unstimulated (−) secretion, or the resulting cell lysates were analyzed by immunoprecipitation with antiinsulin (A) or anti– cathepsin L (B). For INS cells treated with tunicamycin, samples were intentionally double loaded to enhance the sensitivity of detection of mature cathepsin L forms. Note that after tunicamycin treatment, the amount of intracellular precursor increased disproportionately, as did the amount of stimulus-dependent secretion of ProL. The positions of proinsulin, insulin, presumptive proinsulin conversion intermediates (small bracket), glycosylated and unglycosylated ProL, and bands comprising glycosylated and unglycosylated mature cathepsin L (large brackets) are shown.
Mentions: In the experiment shown in Fig. 5, INS cells were pulse labeled and chased for 3.5 h before a 1-h period of stimulus-dependent secretion was analyzed. The left panels show the results from control cells that are similar to Fig. 3, insofar as partial delivery to lysosomes was detected. The right panels were performed in parallel from INS cells treated with tunicamycin. In this case, samples were intentionally double loaded to enhance the sensitivity of detection of mature cathepsin L forms. Nevertheless, it was clear that the arrival of unglycosylated L in lysosomes was profoundly inhibited, while there was a commensurate increase in the amount of intracellular ProL. Evidently, in these cells, despite the low affinity of ProL for MPRs, that portion of ProL which is successfully targeted to lysosomes still uses a carbohydrate-dependent sorting mechanism. Moreover, the increase in missorted ProL after tunicamycin treatment resulted in a proportionate increase in the pool of unglycosylated precursor in the regulated secretory pathway, as demonstrated by markedly increased stimulus-dependent secretion (Fig. 5, right).

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