Biogenesis of the <i>Saccharomyces cerevisiae</i> Mating Pheromone a-Factor

Chen, Peng; Sapperstein, Stephanie K.; Choi, Jonathan; Michaelis, Susan

doi:10.1083/jcb.136.2.251

Cited by 127 publications

(185 citation statements)

References 71 publications

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“…Dried gels were analyzed by autoradiography and with a PhosphorImager. The amino-terminal processing reaction cleaves seven amino acids from the amino terminus of P1, yielding the shorter intermediate P2 (9,29). A, ability of membranes from ES cells and fibroblasts to cleave the P1 a-factor intermediate.…”

Section: Discussionmentioning

confidence: 99%

“…P1 a-factor is fully COOH-terminal modified (i.e. isoprenylated, cleaved, and carboxyl methylated) (29). In a wild-type strain, COOH-terminal processing of a-factor occurs first and is followed by NH 2 -terminal processing (29).…”

Section: Bioassays To Assess Carboxyl-terminal Processing Of An A-facmentioning

confidence: 99%

“…isoprenylated, cleaved, and carboxyl methylated) (29). In a wild-type strain, COOH-terminal processing of a-factor occurs first and is followed by NH 2 -terminal processing (29). Isoprenylation is required (29) for the production of P1 a-factor, but blocking either methylation (30) or CAAX processing 4 does not impede NH 2 -terminal processing.…”

Section: Bioassays To Assess Carboxyl-terminal Processing Of An A-facmentioning

confidence: 99%

“…In a wild-type strain, COOH-terminal processing of a-factor occurs first and is followed by NH 2 -terminal processing (29). Isoprenylation is required (29) for the production of P1 a-factor, but blocking either methylation (30) or CAAX processing 4 does not impede NH 2 -terminal processing. Briefly, a yeast strain lacking STE24 but expressing MFA1 from a high-copy plasmid (SM3103/ pSM219) was radiolabeled with [ 35 S]cysteine, and membranes containing P1 a-factor were isolated (10).…”

Section: Bioassays To Assess Carboxyl-terminal Processing Of An A-facmentioning

confidence: 99%

“…The a-factor intermediates were immunoprecipitated with an a-factor-specific antiserum. Each immunoprecipitate was then size-fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed with a PhosphorImager (10,29).…”

Section: Bioassays To Assess Carboxyl-terminal Processing Of An A-facmentioning

confidence: 99%

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Biochemical Studies of Zmpste24-deficient Mice

Leung¹,

Schmidt²,

Bergö³

et al. 2001

Journal of Biological Chemistry

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100

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Genetic studies in Saccharomyces cerevisiae identified two genes, STE24 and RCE1, involved in cleaving the three carboxyl-terminal amino acids from isoprenylated proteins that terminate with a CAAX sequence motif. Ste24p cleaves the carboxyl-terminal "-AAX" from the yeast mating pheromone a-factor, whereas Rce1p cleaves the -AAX from both a-factor and Ras2p. Ste24p also cleaves the amino terminus of a-factor. The mouse genome contains orthologues for both yeast RCE1 and STE24. We previously demonstrated, with a gene-knockout experiment, that mouse Rce1 is essential for development and that Rce1 is entirely responsible for the carboxyl-terminal proteolytic processing of the mouse Ras proteins. In this study, we cloned mouse Zmpste24, the orthologue for yeast STE24 and showed that it could promote a-factor production when expressed in yeast. Then, to assess the importance of Zmpste24 in development, we generated Zmpste24-deficient mice. Unlike the Rce1 knockout mice, Zmpste24-deficient mice survived development and were fertile. Since no natural substrates for mammalian Zmpste24 have been identified, yeast a-factor was used as a surrogate substrate to investigate the biochemical activities in membranes from the cells and tissues of Zmpste24-deficient mice. We demonstrate that Zmpste24-deficient mouse membranes, like Ste24p-deficient yeast membranes, have diminished CAAX proteolytic activity and lack the ability to cleave the amino terminus of the a-factor precursor. Thus, both enzymatic activities of yeast Ste24p are conserved in mouse Zmpste24, but these enzymatic activities are not essential for mouse development or for fertility.Proteins that terminate in a carboxyl-terminal CAAX motif 1 undergo three sequential enzymatic processing events, farnesylation or geranylgeranylation of the cysteine, endoproteolytic release of the last three amino acid residues of the protein (i.e. removal of the -AAX), and methylation of the new carboxyl terminus of the protein by isoprenylcysteine carboxyl methyltransferase (1, 2). The yeast genes responsible for the farnesylation and methylation steps were identified more than a decade ago (3, 4), but the identification of the genes responsible for the middle processing step, the endoprotease step, remained elusive for years (2). Ultimately, however, Boyartchuk and co-workers (5) applied a novel genetic selection scheme and identified two yeast genes, RCE1 and STE24 (AFC1), involved in the carboxyl-terminal endoproteolytic processing of isoprenylated CAAX proteins. Rce1p is a protease involved in the carboxyl-terminal processing of both a-factor and the yeast Ras protein, Ras2p. Ste24p (Afc1p), a zinc metalloprotease, lacked activity against Ras2p but did process a-factor. Haploid MATa yeast lacking both RCE1 and STE24 (ste24⌬rce1⌬) grew normally but were unable to produce mature a-factor and therefore were sterile (5). Interestingly, Rce1p and Ste24p exhibited subtle differences in substrate specificities (5-7). Both proteins were capable of cleaving the carboxyl terminus of wild-...

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Section: Discussionmentioning

confidence: 99%

Section: Bioassays To Assess Carboxyl-terminal Processing Of An A-facmentioning

confidence: 99%

Section: Bioassays To Assess Carboxyl-terminal Processing Of An A-facmentioning

confidence: 99%

Section: Bioassays To Assess Carboxyl-terminal Processing Of An A-facmentioning

confidence: 99%

Section: Bioassays To Assess Carboxyl-terminal Processing Of An A-facmentioning

confidence: 99%

See 3 more Smart Citations

Biochemical Studies of Zmpste24-deficient Mice

Leung¹,

Schmidt²,

Bergö³

et al. 2001

Journal of Biological Chemistry

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100

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Combining mutations in the incoming and outgoing pheromone signal pathways causes a synergistic mating defect inSaccharomyces cerevisiae

Giot

DeMattei

Konopka

1999

Yeast

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Mating pheromones stimulate Saccharomyces cerevisiae yeast cells to form a pointed projection that becomes the site of cell fusion during conjugation. To investigate the role of mating projections, we screened for mutations that enhanced the weak mating defect of MATa ste2‐T326 cells that are defective in forming pointed projections. These cells are also 10‐fold more sensitive to α‐factor pheromone because ste2‐T326 encodes truncated α‐factor receptors that are not regulated properly. Mutations in AXL1, STE6 and FUS3 were identified in the screen. AXL1 was studied further because it is required for efficient a‐factor pheromone production and for selecting the site for bud morphogenesis. Mutation of AXL1 did not enhance the morphogenesis or pheromone sensitivity defects of ste2‐T326. Instead, the synergistic mating defect was apparently due to decreased a‐factor production because the axl1Δ ste2‐T326 cells mated well with a sst2 α mating partner that is supersensitive to a‐factor. When combined with a wild‐type mating partner, the ste2‐T326 axl1Δ cells failed to mate because they did not lock cell walls, one of the earliest steps in conjugation. Analysis of axl1Δ in combination with other mutations that cause defects in morphogenesis or pheromone sensitivity (e.g. bar1, sst2, afr1) indicated that both phenotypes of ste2‐T326 cells, supersensitivity to α‐factor and the defect in forming pointed projections, contributed to the synergistic mating defect. We suggest a model that the synergistic mating defect is caused by the combined effects of ste2‐T326 and axl1Δ on the presentation of a‐factor to partner cells. Altogether, these results demonstrate an important linkage between the incoming and outgoing pheromone signals during the intercellular communication that promotes yeast mating. Copyright © 1999 John Wiley & Sons, Ltd.

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Identifying (non‐)coding RNAs and small peptides: Challenges and opportunities

2014

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Over the past decade, high-throughput studies have identified many novel transcripts. While their existence is undisputed, their coding potential and functionality have remained controversial. Recent computational approaches guided by ribosome profiling have indicated that translation is far more pervasive than anticipated and takes place on many transcripts previously assumed to be non-coding. Some of these newly discovered translated transcripts encode short, functional proteins that had been missed in prior screens. Other transcripts are translated, but it might be the process of translation rather than the resulting peptides that serve a function. Here, we review annotation studies in zebrafish to discuss the challenges of placing RNAs onto the continuum that ranges from functional protein-encoding mRNAs to potentially non-functional peptide-producing RNAs to non-coding RNAs. As highlighted by the discovery of the novel signaling peptide Apela/ELABELA/Toddler, accurate annotations can give rise to exciting opportunities to identify the functions of previously uncharacterized transcripts.

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Biogenesis of the Saccharomyces cerevisiae Mating Pheromone a-Factor

Cited by 127 publications

References 71 publications

Biochemical Studies of Zmpste24-deficient Mice

Biochemical Studies of Zmpste24-deficient Mice

Combining mutations in the incoming and outgoing pheromone signal pathways causes a synergistic mating defect inSaccharomyces cerevisiae

Identifying (non‐)coding RNAs and small peptides: Challenges and opportunities

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