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Passive sorting in maturing granules of AtT-20 cells: the entry and exit of salivary amylase and proline-rich protein.

Castle AM, Huang AY, Castle JD - J. Cell Biol. (1997)

Bottom Line: Biol.These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage.Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093- 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711- 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.

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Stimulation of secretion of newly synthesized PRP and  amylase. Cells expressing PRP (A) and amylase (B) were labeled for  1 h with 0.5 mCi/ml of [3H]lysine or 0.15 mCi/ml of Expre35S35S  label, respectively. Cells were first chased for 1 or 5 h in the absence of secretagogue, and subsequently for 1 h in the absence  (−) or presence (+) of 5 mM 8-Br-cAMP (8 Br). Immunoprecipitated PRP (A) and amylase (B) from the chases are shown. All  of each immunoprecipitate was loaded except in A for the 5-h  chase, where only 1/3 of the total immunoprecipitate was used.
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Figure 7: Stimulation of secretion of newly synthesized PRP and amylase. Cells expressing PRP (A) and amylase (B) were labeled for 1 h with 0.5 mCi/ml of [3H]lysine or 0.15 mCi/ml of Expre35S35S label, respectively. Cells were first chased for 1 or 5 h in the absence of secretagogue, and subsequently for 1 h in the absence (−) or presence (+) of 5 mM 8-Br-cAMP (8 Br). Immunoprecipitated PRP (A) and amylase (B) from the chases are shown. All of each immunoprecipitate was loaded except in A for the 5-h chase, where only 1/3 of the total immunoprecipitate was used.

Mentions: The similar ER exit rates of PRP and amylase facilitated our analysis of the sorting of newly synthesized exogenous proteins. For this purpose, we monitored the entry and subsequent storage of PRP and amylase in the regulated pathway by applying secretagogues early and late after pulse labeling. Since immature and mature granules are able to undergo stimulus-dependent exocytosis (Arvan et al., 1991; Tooze et al., 1991), early stimulation (1-h chase) tests entry into immature granules, whereas later stimulation (5-h chase) evaluates sustained presence in mature storage granules. This strategy was used previously to demonstrate that lysosomal hydrolase precursors initially enter immature granules in endocrine pancreatic β cells before exiting for lysosomes, whereas proinsulin/insulin enters and remains stably associated with the regulated pathway (Kuliawat and Arvan, 1994). As shown in Fig. 7, A and B, the secretion of both PRP and amylase is clearly stimulated. Although the fold stimulation (1.5 ± 0.1 for PRP and 1.4 ± 0.1 for amylase) is rather low, indicating that there is a high background of unstimulated secretion, these findings clearly indicate that both exogenous proteins enter the regulated pathway. Notably, quantitation (Table II) shows that the percentages of total labeled protein undergoing stimulated secretion (Table II, 8-Br stim.) are considerably higher at 1-h chase than at 5-h chase. For PRP, percent stimulated secretion decreases approximately eightfold between 1 and 5 h of chase (from 10.5 to 1.3%); and for amylase, percent stimulated secretion declines approximately fivefold (from 8% to 1.3%) over the same interval. These changes do not reflect protein degradation as all incorporated label has been accounted for at both time points (Table II, legend). In contrast, the stimulated release of ACTH peptides and of β-endorphin (expressed as percent of total labeled POMC and POMC-derived peptides) assayed under the same conditions was essentially unaffected. Because POMC passes through the early secretory pathway more rapidly than amylase and PRP (t1/2 = 0.5 h; Moore and Kelly, 1985), and thus POMC-related peptides arrive in the immature granule earlier, we also examined stimulability of ACTH peptides using a protocol with better time resolution to confirm that the secretagogue-dependent release is truly constant with time. As shown in Fig. 8, the net stimulated release of ACTH peptides reaches a peak at 1 h of chase and then declines only slightly thereafter. Taken together, these data indicate that both exogenous salivary polypeptides readily enter a stimulatable compartment (immature granules) but are substantially removed during granule maturation. In contrast, POMC-related peptides are largely retained with only minor removal during maturation.


Passive sorting in maturing granules of AtT-20 cells: the entry and exit of salivary amylase and proline-rich protein.

Castle AM, Huang AY, Castle JD - J. Cell Biol. (1997)

Stimulation of secretion of newly synthesized PRP and  amylase. Cells expressing PRP (A) and amylase (B) were labeled for  1 h with 0.5 mCi/ml of [3H]lysine or 0.15 mCi/ml of Expre35S35S  label, respectively. Cells were first chased for 1 or 5 h in the absence of secretagogue, and subsequently for 1 h in the absence  (−) or presence (+) of 5 mM 8-Br-cAMP (8 Br). Immunoprecipitated PRP (A) and amylase (B) from the chases are shown. All  of each immunoprecipitate was loaded except in A for the 5-h  chase, where only 1/3 of the total immunoprecipitate was used.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139952&req=5

Figure 7: Stimulation of secretion of newly synthesized PRP and amylase. Cells expressing PRP (A) and amylase (B) were labeled for 1 h with 0.5 mCi/ml of [3H]lysine or 0.15 mCi/ml of Expre35S35S label, respectively. Cells were first chased for 1 or 5 h in the absence of secretagogue, and subsequently for 1 h in the absence (−) or presence (+) of 5 mM 8-Br-cAMP (8 Br). Immunoprecipitated PRP (A) and amylase (B) from the chases are shown. All of each immunoprecipitate was loaded except in A for the 5-h chase, where only 1/3 of the total immunoprecipitate was used.
Mentions: The similar ER exit rates of PRP and amylase facilitated our analysis of the sorting of newly synthesized exogenous proteins. For this purpose, we monitored the entry and subsequent storage of PRP and amylase in the regulated pathway by applying secretagogues early and late after pulse labeling. Since immature and mature granules are able to undergo stimulus-dependent exocytosis (Arvan et al., 1991; Tooze et al., 1991), early stimulation (1-h chase) tests entry into immature granules, whereas later stimulation (5-h chase) evaluates sustained presence in mature storage granules. This strategy was used previously to demonstrate that lysosomal hydrolase precursors initially enter immature granules in endocrine pancreatic β cells before exiting for lysosomes, whereas proinsulin/insulin enters and remains stably associated with the regulated pathway (Kuliawat and Arvan, 1994). As shown in Fig. 7, A and B, the secretion of both PRP and amylase is clearly stimulated. Although the fold stimulation (1.5 ± 0.1 for PRP and 1.4 ± 0.1 for amylase) is rather low, indicating that there is a high background of unstimulated secretion, these findings clearly indicate that both exogenous proteins enter the regulated pathway. Notably, quantitation (Table II) shows that the percentages of total labeled protein undergoing stimulated secretion (Table II, 8-Br stim.) are considerably higher at 1-h chase than at 5-h chase. For PRP, percent stimulated secretion decreases approximately eightfold between 1 and 5 h of chase (from 10.5 to 1.3%); and for amylase, percent stimulated secretion declines approximately fivefold (from 8% to 1.3%) over the same interval. These changes do not reflect protein degradation as all incorporated label has been accounted for at both time points (Table II, legend). In contrast, the stimulated release of ACTH peptides and of β-endorphin (expressed as percent of total labeled POMC and POMC-derived peptides) assayed under the same conditions was essentially unaffected. Because POMC passes through the early secretory pathway more rapidly than amylase and PRP (t1/2 = 0.5 h; Moore and Kelly, 1985), and thus POMC-related peptides arrive in the immature granule earlier, we also examined stimulability of ACTH peptides using a protocol with better time resolution to confirm that the secretagogue-dependent release is truly constant with time. As shown in Fig. 8, the net stimulated release of ACTH peptides reaches a peak at 1 h of chase and then declines only slightly thereafter. Taken together, these data indicate that both exogenous salivary polypeptides readily enter a stimulatable compartment (immature granules) but are substantially removed during granule maturation. In contrast, POMC-related peptides are largely retained with only minor removal during maturation.

Bottom Line: Biol.These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage.Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.

ABSTRACT
Previous studies have suggested that salivary amylase and proline-rich protein are sorted differently when expressed in AtT-20 cells (Castle, A.M., L.E. Stahl, and J.D. Castle. 1992. J. Biol. Chem. 267:13093- 13100; Colomer, V., K. Lal, T.C. Hoops, and M.J. Rindler. 1994.EMBO (Eur. Mol. Biol. Organ.) J. 13:3711- 3719). We now show that both exocrine proteins behave similarly and enter the regulated secretory pathway as judged by immunolocalization and secretagogue- dependent stimulation of secretion. Analysis of stimulated secretion of newly synthesized proline-rich protein, amylase, and endogenous hormones indicates that the exogenous proteins enter the granule pool with about the same efficiency as the endogenous hormones. However, in contrast to the endogenous hormones, proline-rich protein and amylase are progressively removed from the granule pool during the process of granule maturation such that only small portions remain in mature granules where they colocalize with the stored hormones. The exogenous proteins that are not stored are recovered from the incubation medium and are presumed to have undergone constitutive-like secretion. These results point to a level of sorting for regulated secretion after entry of proteins into forming granules and indicate that retention is essential for efficient storage. Consequently, the critical role of putative sorting receptors for regulated secretion may be in retention rather than in granule entry.

Show MeSH
Related in: MedlinePlus