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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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Secretion of immunoreactive insulin is not mediated by a dominant sorting signal in the α-leader. In this case, the α-leader peptide in ICFP has been replaced by a fragment of the HSP150 leader (as described in Materials and Methods). 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 before analysis by SDS-PAGE and fluorography.
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Figure 8: Secretion of immunoreactive insulin is not mediated by a dominant sorting signal in the α-leader. In this case, the α-leader peptide in ICFP has been replaced by a fragment of the HSP150 leader (as described in Materials and Methods). 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 before analysis by SDS-PAGE and fluorography.

Mentions: All evidence to date indicates that luminal protein targeting from the Golgi complex to the endosomal/vacuolar system requires specific signal recognition, and loss of this sorting results in protein secretion (Marcusson et al. 1994). Nevertheless, we wished to formally exclude the possibility that ICFP secretion might be mediated by a dominant secretion signal in the α-leader peptide and that loss of this signal upon endoproteolytic cleavage might cause single chain insulin to be delivered to the vacuole. Because published evidence indicates that a fragment of the leader peptide from the secretory protein Hsp150 does not contain a dominant secretion signal (Holkeri and Makarow 1998), we replaced the α-leader with this fragment fused to single chain insulin (as described in Materials and Methods). This new insulin-containing chimera was quantitatively secreted (Fig. 8), indicating that secretion of the unprocessed ICFP does not require specific sorting information contained within the α-leader peptide. Thus, intracellular retention of mature insulin cannot be explained by endoproteolytic removal of a dominant secretion signal.


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)

Secretion of immunoreactive insulin is not mediated by a dominant sorting signal in the α-leader. In this case, the α-leader peptide in ICFP has been replaced by a fragment of the HSP150 leader (as described in Materials and Methods). 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 before analysis by SDS-PAGE and fluorography.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Secretion of immunoreactive insulin is not mediated by a dominant sorting signal in the α-leader. In this case, the α-leader peptide in ICFP has been replaced by a fragment of the HSP150 leader (as described in Materials and Methods). 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 before analysis by SDS-PAGE and fluorography.
Mentions: All evidence to date indicates that luminal protein targeting from the Golgi complex to the endosomal/vacuolar system requires specific signal recognition, and loss of this sorting results in protein secretion (Marcusson et al. 1994). Nevertheless, we wished to formally exclude the possibility that ICFP secretion might be mediated by a dominant secretion signal in the α-leader peptide and that loss of this signal upon endoproteolytic cleavage might cause single chain insulin to be delivered to the vacuole. Because published evidence indicates that a fragment of the leader peptide from the secretory protein Hsp150 does not contain a dominant secretion signal (Holkeri and Makarow 1998), we replaced the α-leader with this fragment fused to single chain insulin (as described in Materials and Methods). This new insulin-containing chimera was quantitatively secreted (Fig. 8), indicating that secretion of the unprocessed ICFP does not require specific sorting information contained within the α-leader peptide. Thus, intracellular retention of mature insulin cannot be explained by endoproteolytic removal of a dominant secretion signal.

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