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Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex.

Zhang B, Chang A, Kjeldsen TB, Arvan P - J. Cell Biol. (2001)

Bottom Line: Five out of six eis mutants prove to be vacuolar protein sorting (vps)8, vps35, vps13, vps4, and vps36, which affect Golgi<-->endosome trafficking.Disruption of KEX2, which blocks intracellular processing to insulin, quantitatively reroutes ICFP to the cell surface, whereas loss of the Vps10p sorting receptor is without effect.Secretion of unprocessed ICFP is not based on a dominant secretion signal in the alpha-leader peptide.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
An insulin-containing fusion protein (ICFP, encoding the yeast prepro-alpha factor leader peptide fused via a lysine-arginine cleavage site to a single chain insulin) has been expressed in Saccharomyces cerevisiae where it is inefficiently secreted. Single gene disruptions have been identified that cause enhanced immunoreactive insulin secretion (eis). Five out of six eis mutants prove to be vacuolar protein sorting (vps)8, vps35, vps13, vps4, and vps36, which affect Golgi<-->endosome trafficking. Indeed, in wild-type yeast insulin is ultimately delivered to the vacuole, whereas vps mutants secrete primarily unprocessed ICFP. Disruption of KEX2, which blocks intracellular processing to insulin, quantitatively reroutes ICFP to the cell surface, whereas loss of the Vps10p sorting receptor is without effect. Secretion of unprocessed ICFP is not based on a dominant secretion signal in the alpha-leader peptide. Although insulin sorting mediated by Kex2p is saturable, Kex2p functions not as a sorting receptor but as a protease: replacement of Kex2p by truncated secretory Kex2p (which travels from Golgi to cell surface) still causes endoproteolytic processing and intracellular insulin retention. Endoproteolysis promotes a change in insulin's biophysical properties. B5His residues normally participate in multimeric insulin packing; a point mutation at this position permits ICFP processing but causes the majority of processed insulin to be secreted. The data argue that multimeric assembly consequent to endoproteolytic maturation regulates insulin sorting in the secretory pathway.

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The ICFP is not subject to ERAD but is subject to degradation by vacuolar proteases. (A) The ICFP was expressed from the GPD1 promoter in a sec18-1 mutant strain where the ICFP cannot reach the Golgi complex. Cells were preincubated at the restrictive temperature (37°C) for 10 min and labeled for 5 min. Cells were chased for the times indicated, lysed, and immunoprecipitated with antiinsulin and digested with PNGase F before SDS-PAGE. (B) Quantification by densitometry of the results shown in A. (C) The ICFP was expressed in a pep4- strain. The cells were labeled and chased as in the legend to Fig. 2 B before insulin immunoprecipitation and digestion with PNGaseF followed by SDS-PAGE and fluorography. Note that in a sec18 strain intracellular ICFP persists, whereas in a pep4 strain intracellular insulin accumulates.
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Figure 4: The ICFP is not subject to ERAD but is subject to degradation by vacuolar proteases. (A) The ICFP was expressed from the GPD1 promoter in a sec18-1 mutant strain where the ICFP cannot reach the Golgi complex. Cells were preincubated at the restrictive temperature (37°C) for 10 min and labeled for 5 min. Cells were chased for the times indicated, lysed, and immunoprecipitated with antiinsulin and digested with PNGase F before SDS-PAGE. (B) Quantification by densitometry of the results shown in A. (C) The ICFP was expressed in a pep4- strain. The cells were labeled and chased as in the legend to Fig. 2 B before insulin immunoprecipitation and digestion with PNGaseF followed by SDS-PAGE and fluorography. Note that in a sec18 strain intracellular ICFP persists, whereas in a pep4 strain intracellular insulin accumulates.

Mentions: Even though enhanced immunoreactive insulin secretion was observed in several vps mutants acting in the distal secretory pathway, ER to Golgi transport is known to be a rate-limiting step in the trafficking of chimeras containing the prepro-α leader (Elliott et al. 1989). We therefore employed a standard assay (Finger et al. 1993; Loayza et al. 1998) to examine whether ICFP is subject to ERAD. The ICFP was expressed in a sec18 strain in which traffic to the Golgi complex is blocked at the nonpermissive temperature. After shifting to 37°C, cells were pulse labeled, lysed at varying times of chase ≤1.5 h, and analyzed by immunoprecipitation with antiinsulin, PNGase F digestion, and tricine-urea–SDS-PAGE. Strikingly, no endoproteolytic processing of ICFP was observed in the sec18 background (Fig. 4A and Fig. B) in contrast to wild-type yeast (see Fig. 2 A). Furthermore, unlike what has been observed for ERAD substrates (Plemper and Wolf 1999) the ICFP molecule was completely stable throughout the 90-min time course. Thus, no evidence was obtained for ERAD of the insulin-containing chimera.


Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex.

Zhang B, Chang A, Kjeldsen TB, Arvan P - J. Cell Biol. (2001)

The ICFP is not subject to ERAD but is subject to degradation by vacuolar proteases. (A) The ICFP was expressed from the GPD1 promoter in a sec18-1 mutant strain where the ICFP cannot reach the Golgi complex. Cells were preincubated at the restrictive temperature (37°C) for 10 min and labeled for 5 min. Cells were chased for the times indicated, lysed, and immunoprecipitated with antiinsulin and digested with PNGase F before SDS-PAGE. (B) Quantification by densitometry of the results shown in A. (C) The ICFP was expressed in a pep4- strain. The cells were labeled and chased as in the legend to Fig. 2 B before insulin immunoprecipitation and digestion with PNGaseF followed by SDS-PAGE and fluorography. Note that in a sec18 strain intracellular ICFP persists, whereas in a pep4 strain intracellular insulin accumulates.
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Related In: Results  -  Collection

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Figure 4: The ICFP is not subject to ERAD but is subject to degradation by vacuolar proteases. (A) The ICFP was expressed from the GPD1 promoter in a sec18-1 mutant strain where the ICFP cannot reach the Golgi complex. Cells were preincubated at the restrictive temperature (37°C) for 10 min and labeled for 5 min. Cells were chased for the times indicated, lysed, and immunoprecipitated with antiinsulin and digested with PNGase F before SDS-PAGE. (B) Quantification by densitometry of the results shown in A. (C) The ICFP was expressed in a pep4- strain. The cells were labeled and chased as in the legend to Fig. 2 B before insulin immunoprecipitation and digestion with PNGaseF followed by SDS-PAGE and fluorography. Note that in a sec18 strain intracellular ICFP persists, whereas in a pep4 strain intracellular insulin accumulates.
Mentions: Even though enhanced immunoreactive insulin secretion was observed in several vps mutants acting in the distal secretory pathway, ER to Golgi transport is known to be a rate-limiting step in the trafficking of chimeras containing the prepro-α leader (Elliott et al. 1989). We therefore employed a standard assay (Finger et al. 1993; Loayza et al. 1998) to examine whether ICFP is subject to ERAD. The ICFP was expressed in a sec18 strain in which traffic to the Golgi complex is blocked at the nonpermissive temperature. After shifting to 37°C, cells were pulse labeled, lysed at varying times of chase ≤1.5 h, and analyzed by immunoprecipitation with antiinsulin, PNGase F digestion, and tricine-urea–SDS-PAGE. Strikingly, no endoproteolytic processing of ICFP was observed in the sec18 background (Fig. 4A and Fig. B) in contrast to wild-type yeast (see Fig. 2 A). Furthermore, unlike what has been observed for ERAD substrates (Plemper and Wolf 1999) the ICFP molecule was completely stable throughout the 90-min time course. Thus, no evidence was obtained for ERAD of the insulin-containing chimera.

Bottom Line: Five out of six eis mutants prove to be vacuolar protein sorting (vps)8, vps35, vps13, vps4, and vps36, which affect Golgi<-->endosome trafficking.Disruption of KEX2, which blocks intracellular processing to insulin, quantitatively reroutes ICFP to the cell surface, whereas loss of the Vps10p sorting receptor is without effect.Secretion of unprocessed ICFP is not based on a dominant secretion signal in the alpha-leader peptide.

View Article: PubMed Central - PubMed

Affiliation: Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.

ABSTRACT
An insulin-containing fusion protein (ICFP, encoding the yeast prepro-alpha factor leader peptide fused via a lysine-arginine cleavage site to a single chain insulin) has been expressed in Saccharomyces cerevisiae where it is inefficiently secreted. Single gene disruptions have been identified that cause enhanced immunoreactive insulin secretion (eis). Five out of six eis mutants prove to be vacuolar protein sorting (vps)8, vps35, vps13, vps4, and vps36, which affect Golgi<-->endosome trafficking. Indeed, in wild-type yeast insulin is ultimately delivered to the vacuole, whereas vps mutants secrete primarily unprocessed ICFP. Disruption of KEX2, which blocks intracellular processing to insulin, quantitatively reroutes ICFP to the cell surface, whereas loss of the Vps10p sorting receptor is without effect. Secretion of unprocessed ICFP is not based on a dominant secretion signal in the alpha-leader peptide. Although insulin sorting mediated by Kex2p is saturable, Kex2p functions not as a sorting receptor but as a protease: replacement of Kex2p by truncated secretory Kex2p (which travels from Golgi to cell surface) still causes endoproteolytic processing and intracellular insulin retention. Endoproteolysis promotes a change in insulin's biophysical properties. B5His residues normally participate in multimeric insulin packing; a point mutation at this position permits ICFP processing but causes the majority of processed insulin to be secreted. The data argue that multimeric assembly consequent to endoproteolytic maturation regulates insulin sorting in the secretory pathway.

Show MeSH
Related in: MedlinePlus