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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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Deletion of KEX2 causes near quantitative rerouting of ICFP to the medium. The strains are pep4Δ mutants in order to stabilize the intracellular protein. Cells were pulse labeled for 30 min and chased for 60 min. The lysed cells and media underwent insulin immunoprecipitation and digestion with PNGaseF before analysis by SDS-PAGE and fluorography.
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Figure 7: Deletion of KEX2 causes near quantitative rerouting of ICFP to the medium. The strains are pep4Δ mutants in order to stabilize the intracellular protein. Cells were pulse labeled for 30 min and chased for 60 min. The lysed cells and media underwent insulin immunoprecipitation and digestion with PNGaseF before analysis by SDS-PAGE and fluorography.

Mentions: Therefore, we reexamined ICFP expression (driven from a GAL1 promoter on a centromeric plasmid) in a kex2 pep4 strain. As demonstrated before, in the presence of wild-type levels of Kex2p, virtually all ICFP was processed to single chain insulin, and only a small fraction (<10%) was secreted (Fig. 7, left). However, in the absence of Kex2p not only was there no ICFP cleavage, but the protein was near quantitatively rerouted to the medium (Fig. 7, right).


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)

Deletion of KEX2 causes near quantitative rerouting of ICFP to the medium. The strains are pep4Δ mutants in order to stabilize the intracellular protein. Cells were pulse labeled for 30 min and chased for 60 min. The lysed cells and media underwent insulin immunoprecipitation and digestion with PNGaseF before analysis by SDS-PAGE and fluorography.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2192022&req=5

Figure 7: Deletion of KEX2 causes near quantitative rerouting of ICFP to the medium. The strains are pep4Δ mutants in order to stabilize the intracellular protein. Cells were pulse labeled for 30 min and chased for 60 min. The lysed cells and media underwent insulin immunoprecipitation and digestion with PNGaseF before analysis by SDS-PAGE and fluorography.
Mentions: Therefore, we reexamined ICFP expression (driven from a GAL1 promoter on a centromeric plasmid) in a kex2 pep4 strain. As demonstrated before, in the presence of wild-type levels of Kex2p, virtually all ICFP was processed to single chain insulin, and only a small fraction (<10%) was secreted (Fig. 7, left). However, in the absence of Kex2p not only was there no ICFP cleavage, but the protein was near quantitatively rerouted to the medium (Fig. 7, right).

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