Yeast as a Tractable Genetic System for Functional Studies of the Insulin-degrading Enzyme

Kim, Seonil; Lapham, Andrea N.; Freedman, Christopher G.K.; Reed, Tiffany L.; Schmidt, W.

doi:10.1074/jbc.m414192200

Cited by 38 publications

(59 citation statements)

References 45 publications

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“…Ste23 also possesses an HXXEH motif. Both Axl1 and Ste23 are members of the M16A subfamily of zinc metalloproteases (140,186).…”

Section: Axl1 Is a Predicted Zinc Metalloprotease That Generates Matumentioning

confidence: 99%

“…Interest in this enzyme has grown, as IDE deficiency correlates with an increased risk for type 2 diabetes and Alzheimer's disease, although the physiological importance of IDE1 for human health and disease remains to be firmly established (159). Mammalian IDE heterologously expressed in yeast can substitute for Axl1 to mediate a-factor biogenesis (140). Thus, yeast may provide a tractable model system for studying the functional properties of human IDE1.…”

Section: Axl1 Is a Predicted Zinc Metalloprotease That Generates Matumentioning

confidence: 99%

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Biogenesis of the Saccharomyces cerevisiae Pheromone a -Factor, from Yeast Mating to Human Disease

Michaelis¹,

Barrowman²

2012

Microbiol Mol Biol Rev

106

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SUMMARY The mating pheromone a -factor secreted by Saccharomyces cerevisiae is a farnesylated and carboxylmethylated peptide and is unusually hydrophobic compared to other extracellular signaling molecules. Mature a -factor is derived from a precursor with a C-terminal CAAX motif that directs a series of posttranslational reactions, including prenylation, endoproteolysis, and carboxylmethylation. Historically, a -factor has served as a valuable model for the discovery and functional analysis of CAAX-processing enzymes. In this review, we discuss the three modules comprising the a -factor biogenesis pathway: (i) the C-terminal CAAX-processing steps carried out by Ram1/Ram2, Ste24 or Rce1, and Ste14; (ii) two sequential N-terminal cleavage steps, mediated by Ste24 and Axl1; and (iii) export by a nonclassical mechanism, mediated by the ATP binding cassette (ABC) transporter Ste6. The small size and hydrophobicity of a -factor present both challenges and advantages for biochemical analysis, as discussed here. The enzymes involved in a -factor biogenesis are conserved from yeasts to mammals. Notably, studies of the zinc metalloprotease Ste24 in S. cerevisiae led to the discovery of its mammalian homolog ZMPSTE24, which cleaves the prenylated C-terminal tail of the nuclear scaffold protein lamin A. Mutations that alter ZMPSTE24 processing of lamin A in humans cause the premature-aging disease progeria and related progeroid disorders. Intriguingly, recent evidence suggests that the entire a -factor pathway, including all three biogenesis modules, may be used to produce a prenylated, secreted signaling molecule involved in germ cell migration in Drosophila . Thus, additional prenylated signaling molecules resembling a -factor, with as-yet-unknown roles in metazoan biology, may await discovery.

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“…Ste23 also possesses an HXXEH motif. Both Axl1 and Ste23 are members of the M16A subfamily of zinc metalloproteases (140,186).…”

Section: Axl1 Is a Predicted Zinc Metalloprotease That Generates Matumentioning

confidence: 99%

Section: Axl1 Is a Predicted Zinc Metalloprotease That Generates Matumentioning

confidence: 99%

Biogenesis of the Saccharomyces cerevisiae Pheromone a -Factor, from Yeast Mating to Human Disease

Michaelis¹,

Barrowman²

2012

Microbiol Mol Biol Rev

106

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“…IDE Activity Assay in Yeast-Wild type IDE and IDE K898A,K899A,S901A activities were compared in Saccharomyces cerevisiae using both a serial dilution mating test and a spot halo assay as described previously (37,38). The assays take advantage of the ability of IDE, in lieu of the yeast M16 metallopeptidases Axl1p and Ste23p, to participate in the proteolytic (40)).…”

Section: Preparation Of Ide Mutants-ratmentioning

confidence: 99%

“…Effect of ATP-binding Site Mutation on Intracellular Activity of IDE-An established yeast system for functional studies of IDE (37,38) was used to assess the significance of anion activation on IDE activity in a cellular environment. The assay is based on the ability of IDE to support production of mature a-factor mating pheromone in the absence of yeast metallopeptidases (i.e.…”

Section: Ide-atp Complex Crystal Structure-previous Studies Havementioning

confidence: 99%

Anion Activation Site of Insulin-degrading Enzyme

Noinaj

Song

Bhasin

et al. 2012

Journal of Biological Chemistry

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Insulin-degrading enzyme (IDE) (insulysin) is a zinc metallopeptidase that metabolizes several bioactive peptides, including insulin and the amyloid ␤ peptide. IDE is an unusual metallopeptidase in that it is allosterically activated by both small peptides and anions, such as ATP. Here, we report that the ATPbinding site is located on a portion of the substrate binding chamber wall arising largely from domain 4 of the four-domain IDE. Two variants having residues in this site mutated, IDE K898A,K899A,S901A and IDE R429S , both show greatly decreased activation by the polyphosphate anions ATP and PPP i . IDE K898A,K899A,S901A is also deficient in activation by small peptides, suggesting a possible mechanistic link between the two types of allosteric activation. Sodium chloride at high concentrations can also activate IDE. There are no observable differences in average conformation between the IDE-ATP complex and unliganded IDE, but regions of the active site and C-terminal domain do show increased crystallographic thermal factors in the complex, suggesting an effect on dynamics. Activation by ATP is shown to be independent of the ATP hydrolysis activity reported for the enzyme. We also report that IDE K898A,K899A,S901A has reduced intracellular function relative to unmodified IDE, consistent with a possible role for anion activation of IDE activity in vivo. Together, the data suggest a model in which the binding of anions activates by reducing the electrostatic attraction between the two halves of the enzyme, shifting the partitioning between open and closed conformations of IDE toward the open form.

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“…The second stage in a-factor maturation involves cleavage of the N-terminal extension in two sequential steps mediated by Ste24p (step 4, P1 3 P2) (28,64) and Axl1p (step 5, P2 3 M) (1). Ste23p (not shown) acts redundantly with Axl1p, but its contribution to processing is minor (1,35). Finally, export of a-factor out of the cell is mediated by an ATP binding cassette transporter, Ste6p (step 6) (5,37,42,43).…”

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confidence: 99%

Saccharomyces cerevisiae a -Factor Mutants Reveal Residues Critical for Processing, Activity, and Export

et al. 2006

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The Saccharomyces cerevisiae mating pheromone a-factor provides a paradigm for understanding the biogenesis of prenylated fungal pheromones. The biogenesis of a-factor involves multiple steps: (i) C-terminal CAAX modification (where C is cysteine, A is aliphatic, and X is any residue) which includes prenylation, proteolysis, and carboxymethylation (by Ram1p/Ram2p, Ste24p or Rce1p, and Ste14p, respectively); (ii) N-terminal processing, involving two sequential proteolytic cleavages (by Ste24p and Axl1p); and (iii) nonclassical export (by Ste6p). Once exported, mature a-factor interacts with the Ste3p receptor on MAT␣ cells to stimulate mating. The a-factor biogenesis machinery is well defined, as is the CAAX motif that directs C-terminal modification; however, very little is known about the sequence determinants within a-factor required for N-terminal processing, activity, and export. Here we generated a large collection of a-factor mutants and identified residues critical for the N-terminal processing steps mediated by Ste24p and Axl1p. We also identified mutants that fail to support mating but do not affect biogenesis or export, suggesting a defective interaction with the Ste3p receptor. Mutants significantly impaired in export were also found, providing evidence that the Ste6p transporter recognizes sequence determinants as well as CAAX modifications. We also performed a phenotypic analysis of the entire set of isogenic a-factor biogenesis machinery mutants, which revealed information about the dependency of biogenesis steps upon one another, and demonstrated that export by Ste6p requires the completion of all processing events. Overall, this comprehensive analysis will provide a useful framework for the study of other fungal pheromones, as well as prenylated metazoan proteins involved in development and aging.Most fungi secrete pheromones that play important signaling roles in mating to stimulate cell and/or nuclear fusion. In Saccharomyces cerevisiae and other ascomycetes, the mating pheromones produced by the two mating types differ, in that one is an unmodified peptide, while the other is a prenylated and carboxymethylated peptide (i.e., ␣-factor and a-factor, respectively) (3,7,21,24,25,41,49,59,61,70). Basidomycetes, which can have two or even more mating types, encode exclusively the latter class of modified peptide pheromones (8,13,18,26,27,36,47,56,60). These pheromones are synthesized as precursors terminating in a C-terminal CAAX motif (where C is cysteine, A is usually aliphatic, and X is any residue). The S. cerevisiae pheromone a-factor is the best-characterized example of this class of fungal pheromones and provides a paradigm for understanding their biogenesis and secretion (14,20). Studies of a-factor have also yielded critical insights into the processing and properties of eukaryotic CAAX proteins in general, which include Ras and other small GTP-binding proteins, the ␥-subunits of heterotrimeric G proteins, and the nuclear lamins A and B (30,68,69).Components of the machinery required for th...

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Yeast as a Tractable Genetic System for Functional Studies of the Insulin-degrading Enzyme

Cited by 38 publications

References 45 publications

Biogenesis of the Saccharomyces cerevisiae Pheromone a -Factor, from Yeast Mating to Human Disease

Biogenesis of the Saccharomyces cerevisiae Pheromone a -Factor, from Yeast Mating to Human Disease

Anion Activation Site of Insulin-degrading Enzyme

Saccharomyces cerevisiae a -Factor Mutants Reveal Residues Critical for Processing, Activity, and Export

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