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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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Identification and characterization of eis mutants that yield enhanced insulin secretion. (A) Filter blot assay for immunoreactive insulin secretion from the nine colonies obtained by genetic screening, which represent disruptions of six independent genes. Five of these proved to be genes in the vps pathway as indicated. (B) Five eis/vps mutants and a vps10- mutant were replica plated and tested by filter blot assay for immunoreactive insulin secretion (top panel) and CPY secretion (bottom panel). Note that like the eis/vps mutants obtained by genetic screen vps10 dramatically increases CPY secretion, but the effect on ICFP secretion is minimal.
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Figure 3: Identification and characterization of eis mutants that yield enhanced insulin secretion. (A) Filter blot assay for immunoreactive insulin secretion from the nine colonies obtained by genetic screening, which represent disruptions of six independent genes. Five of these proved to be genes in the vps pathway as indicated. (B) Five eis/vps mutants and a vps10- mutant were replica plated and tested by filter blot assay for immunoreactive insulin secretion (top panel) and CPY secretion (bottom panel). Note that like the eis/vps mutants obtained by genetic screen vps10 dramatically increases CPY secretion, but the effect on ICFP secretion is minimal.

Mentions: The foregoing results suggested some form of quality control of insulin secretion. To examine this further, a genetic approach was undertaken. Using yeast expressing ICFP from a single copy integrated in the genome (see Fig. 1 B) and a mutagenized genomic library containing random insertions of lacZ and LEU2 (Burns et al. 1994), 90,000 transformants were screened by the Western blot assay to yield nine eis mutants displaying enhanced immunoreactive insulin secretion (as described in Materials and Methods). The nine eis mutants corresponded to six distinct gene disruptions, five of which were identified as VPS35, VPS13, VPS4, VPS8, and VPS36. These genes have been shown previously to regulate trafficking in the Golgi↔endosome↔vacuole pathway (Stack and Emr 1993; Bryant and Stevens 1998). vps35 and vps8 appeared to produce the greatest enhancement of immunoreactive insulin secretion, vps4 and vps13 produced an intermediate enhancement, and vps36 produced only a slight increase (Fig. 3 A).


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)

Identification and characterization of eis mutants that yield enhanced insulin secretion. (A) Filter blot assay for immunoreactive insulin secretion from the nine colonies obtained by genetic screening, which represent disruptions of six independent genes. Five of these proved to be genes in the vps pathway as indicated. (B) Five eis/vps mutants and a vps10- mutant were replica plated and tested by filter blot assay for immunoreactive insulin secretion (top panel) and CPY secretion (bottom panel). Note that like the eis/vps mutants obtained by genetic screen vps10 dramatically increases CPY secretion, but the effect on ICFP secretion is minimal.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Identification and characterization of eis mutants that yield enhanced insulin secretion. (A) Filter blot assay for immunoreactive insulin secretion from the nine colonies obtained by genetic screening, which represent disruptions of six independent genes. Five of these proved to be genes in the vps pathway as indicated. (B) Five eis/vps mutants and a vps10- mutant were replica plated and tested by filter blot assay for immunoreactive insulin secretion (top panel) and CPY secretion (bottom panel). Note that like the eis/vps mutants obtained by genetic screen vps10 dramatically increases CPY secretion, but the effect on ICFP secretion is minimal.
Mentions: The foregoing results suggested some form of quality control of insulin secretion. To examine this further, a genetic approach was undertaken. Using yeast expressing ICFP from a single copy integrated in the genome (see Fig. 1 B) and a mutagenized genomic library containing random insertions of lacZ and LEU2 (Burns et al. 1994), 90,000 transformants were screened by the Western blot assay to yield nine eis mutants displaying enhanced immunoreactive insulin secretion (as described in Materials and Methods). The nine eis mutants corresponded to six distinct gene disruptions, five of which were identified as VPS35, VPS13, VPS4, VPS8, and VPS36. These genes have been shown previously to regulate trafficking in the Golgi↔endosome↔vacuole pathway (Stack and Emr 1993; Bryant and Stevens 1998). vps35 and vps8 appeared to produce the greatest enhancement of immunoreactive insulin secretion, vps4 and vps13 produced an intermediate enhancement, and vps36 produced only a slight increase (Fig. 3 A).

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