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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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Model of ICFP and insulin sorting in the secretory pathway of yeast cells. According to this schema, ICFP is quantitatively delivered to the Golgi complex where it may encounter Kex2p. To the extent that the precursor protein is unprocessed by Kex2p, it is delivered to the cell surface via the secretory pathway. To the extent that ICFP is endoproteolytically processed in the Golgi complex, insulin is retained intracellularly and is delivered ultimately to the vacuole. Note that the α-leader peptide contains no [35S]cysteine residues; thus, we have not analyzed the fate of the α-leader peptide after cleavage from the insulin moiety.
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Figure 12: Model of ICFP and insulin sorting in the secretory pathway of yeast cells. According to this schema, ICFP is quantitatively delivered to the Golgi complex where it may encounter Kex2p. To the extent that the precursor protein is unprocessed by Kex2p, it is delivered to the cell surface via the secretory pathway. To the extent that ICFP is endoproteolytically processed in the Golgi complex, insulin is retained intracellularly and is delivered ultimately to the vacuole. Note that the α-leader peptide contains no [35S]cysteine residues; thus, we have not analyzed the fate of the α-leader peptide after cleavage from the insulin moiety.

Mentions: It has been reported that improved secretion efficiency of some insulin-containing chimeras can be achieved by replacing an α-leader peptide with synthetic leader sequences that alter ER residence time, suggesting that ER quality control is an important feature of insulin secretion in yeast (Kjeldsen et al. 1997). However, we have discovered several additional features of the system not typical of ER quality control. First, using the ICFP construct described in Fig. 1 A, simple overexpression caused significantly enhanced secretion of immunoreactive insulin (Fig. 1 C), suggesting that one or more saturable components in the yeast secretory pathway limits insulin secretion. Second, newly-synthesized ICFP, containing a single dibasic cleavage site, was rapidly processed to a band comigrating with authentic insulin (Fig. 2 A) in a manner dependent on arrival in the Golgi complex (Fig. 4 A) and Kex2p activity (Fig. 7). Third, a direct assay for ERAD (in a sec18 mutant) showed ICFP to be completely stable over a 90-min time course (Fig. 4A and Fig. B), suggesting that it is not a ready substrate for typical ubiquitin-mediated proteasome-dependent proteolysis (Plemper and Wolf 1999). Fourth, in the absence of Pep4p (vacuolar proteinase A) insulin accumulates not in the ER but in the vacuole (Fig. 4 C) where it is delivered via the endosomal system (Fig. 5). These novel features of ICFP and insulin sorting are summarized in Fig. 12.


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)

Model of ICFP and insulin sorting in the secretory pathway of yeast cells. According to this schema, ICFP is quantitatively delivered to the Golgi complex where it may encounter Kex2p. To the extent that the precursor protein is unprocessed by Kex2p, it is delivered to the cell surface via the secretory pathway. To the extent that ICFP is endoproteolytically processed in the Golgi complex, insulin is retained intracellularly and is delivered ultimately to the vacuole. Note that the α-leader peptide contains no [35S]cysteine residues; thus, we have not analyzed the fate of the α-leader peptide after cleavage from the insulin moiety.
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Related In: Results  -  Collection

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Figure 12: Model of ICFP and insulin sorting in the secretory pathway of yeast cells. According to this schema, ICFP is quantitatively delivered to the Golgi complex where it may encounter Kex2p. To the extent that the precursor protein is unprocessed by Kex2p, it is delivered to the cell surface via the secretory pathway. To the extent that ICFP is endoproteolytically processed in the Golgi complex, insulin is retained intracellularly and is delivered ultimately to the vacuole. Note that the α-leader peptide contains no [35S]cysteine residues; thus, we have not analyzed the fate of the α-leader peptide after cleavage from the insulin moiety.
Mentions: It has been reported that improved secretion efficiency of some insulin-containing chimeras can be achieved by replacing an α-leader peptide with synthetic leader sequences that alter ER residence time, suggesting that ER quality control is an important feature of insulin secretion in yeast (Kjeldsen et al. 1997). However, we have discovered several additional features of the system not typical of ER quality control. First, using the ICFP construct described in Fig. 1 A, simple overexpression caused significantly enhanced secretion of immunoreactive insulin (Fig. 1 C), suggesting that one or more saturable components in the yeast secretory pathway limits insulin secretion. Second, newly-synthesized ICFP, containing a single dibasic cleavage site, was rapidly processed to a band comigrating with authentic insulin (Fig. 2 A) in a manner dependent on arrival in the Golgi complex (Fig. 4 A) and Kex2p activity (Fig. 7). Third, a direct assay for ERAD (in a sec18 mutant) showed ICFP to be completely stable over a 90-min time course (Fig. 4A and Fig. B), suggesting that it is not a ready substrate for typical ubiquitin-mediated proteasome-dependent proteolysis (Plemper and Wolf 1999). Fourth, in the absence of Pep4p (vacuolar proteinase A) insulin accumulates not in the ER but in the vacuole (Fig. 4 C) where it is delivered via the endosomal system (Fig. 5). These novel features of ICFP and insulin sorting are summarized in Fig. 12.

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