Articles

Activation of the SPS Amino Acid-Sensing Pathway in Saccharomyces cerevisiae Correlates with the Phosphorylation State of a Sensor Component, Ptr3

Zhengchang Liu, Janet Thornton, Mário Spírek, Ronald A. Butow
DOI: 10.1128/MCB.00929-07

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    Ptr3 is a phosphoprotein. (A) A 6× myc-tagged Ptr3 is functional. A ptr3Δ deletion strain with an integrated AGP1-lacZ reporter gene (ZLY1917) was transformed with PTR3 (pZL473), vector (pRS416), or PTR3-myc (pZL1179) plasmid as indicated. Transformants were grown in SD medium with or without 0.02% leucine, and β-galactosidase activities were conducted as described in Materials and Methods. (B) Ptr3 is phosphorylated. The ptr3Δ strain transformed with PTR3 (pZL473) or PTR3-myc (pZL1179) plasmids used as described for panel A was grown in SD medium with 0.02% leucine to mid-log phase. TCA-precipitated total cellular proteins were treated with lambda protein phosphatase (λ PPase) as indicated. When required, phosphatase inhibitors (see Materials and Methods) were added to inhibit λ PPase. After treatment, proteins were separated by SDS-PAGE and Ptr3-myc was probed with anti-myc antibody. Arrows and the arrowhead indicate phosphorylated and dephosphorylated forms of Ptr3-myc, respectively. (C) Ptr3 hyperphosphorylation is blocked in ssy1Δ cells but not in ssy5Δ or grr1Δ cells. Yeast strains (ptr3Δ, HKY31; ssy1Δ ptr3Δ, RBY923; ssy5Δ ptr3Δ, RBY875, ssy1Δ ssy5Δ ptr3Δ, RBY951; grr1Δ, ZLY175) transformed with a centromeric plasmid carrying either PTR3 (no tag; pZL473) or PTR3-myc (pZL1179) as indicated were grown in SD medium supplemented with 0.02% leucine. Ptr3-myc was probed with anti-myc antibody. “hyperphos,” “hypophos,” and “dephos” indicate hyperphosphorylated, hypophosphorylated, and dephosphorylated forms of Ptr3-myc, respectively. (D) Leucine-induced hyperphosphorylation of Ptr3 requires Grr1. A wild-type (WT) strain and a grr1Δ mutant strain carrying an integrated copy of PTR3-myc (WT, ZLY2142; grr1Δ, ZLY2916) were grown in SD medium with or without 0.02% leucine. Total cellular proteins from equal amounts of cells were separated by SDS-PAGE and Ptr3-myc was probed with anti-myc antibody. The numbers (average from three independent experiments) indicate the ratios of the levels of hyperphosphorylated and hypophosphorylated forms of Ptr3-myc.

  • FIG. 2.
    FIG. 2.

    Yck1 and Yck2, two CKI proteins, are required for the hyperphosphorylation of Ptr3. (A) The hyperphosphorylation of Ptr3 is blocked in yck1Δ yck2ts double mutant cells. Wild-type (WT; LRB341), yck1Δ (ZLY2021), yck2Δ (LRB343), and yck1Δ yck2ts (LRB362) mutant cells expressing either nontagged Ptr3 (pZL473) or Ptr3-myc from a centromeric plasmid (pZL1179) were grown at the indicated temperatures (Temp) in SD medium supplemented with 0.02% leucine. Total cellular proteins were separated by SDS-PAGE and Ptr3-myc was probed with anti-myc antibody. “hyperphos” and “hypophos” indicate the hyperphosphorylated and hypophosphorylated forms of Ptr3-myc, respectively. (B) The yck1Δ yck2ts double mutation blocks proteolytic processing of Stp1. Wild-type (LRB341) and yck1Δ yck2ts mutant (LRB362) cells transformed with a centromeric plasmid carrying STP1-HA (pZL1834) were grown at the indicated temperatures in SD medium supplemented with 0.02% leucine. Total cellular proteins were separated by SDS-PAGE and Stp1-HA was probed with anti-HA antibody. The full-length Stp1-HA and the processed Stp1-HA are indicated diagrammatically in the figure.

  • FIG. 3.
    FIG. 3.

    Mutations in threonine residue 525 abolish the hyperphosphorylation of Ptr3 and inactivate Ptr3. (A) A schematic representation of CKI consensus sites and a conserved zinc finger motif in Ptr3. (B) β-Galactosidase activity assay of AGP1-lacZ reporter gene expression in ptr3Δ cells transformed with various PTR3 constructs. ptr3Δ cells (ZLY1917) were transformed with a plasmid carrying wild-type PTR3 or mutant ptr3 alleles as indicated [PTR3, pZL1949; PTR3(S321A), pZL2107; PTR3(T635A), pZL2112; PTR3(T525A), pZL2122; PTR3(T525D), pZL2125; PTR3(T525E), pZL2132]. The resultant transformants were grown in SD medium with or without 0.02% leucine, and β-galactosidase assays were conducted as described previously. (C) Mutations in threonine 525 block the hyperphosphorylation of Ptr3. Cells used for panel B were grown in SD medium supplemented with 0.02% leucine. Total cellular proteins were separated by SDS-PAGE. Wild-type Ptr3-myc and various mutant Ptr3-myc proteins were probed with anti-myc antibody. “hyperphos,” “hypophos,” and “dephos” indicate hyperphosphorylated, hypophosphorylated, and dephosphorylated forms of Ptr3-myc, respectively. (D) Blocks in Ptr3 hyperphosphorylation correlate with a failure to process Stp1. ptr3Δ cells (HKY31) carrying centromeric plasmids coexpressing Stp1-HA (pZL2230) and various PTR3 constructs as described for panel B were grown in SD medium with or without 0.02% leucine as indicated. Total cellular proteins were separated by SDS-PAGE and Stp1-HA was probed with anti-HA antibody. The full-length Stp1-HA and the processed Stp1-HA are indicated diagrammatically in the figure.

  • FIG. 4.
    FIG. 4.

    An activating mutant allele of PTR3, PTR3(Q439), leads to Ptr3 hyperphosphorylation. (A) A PTR3(Q439R) mutation, due to a change of residue 439 from glutamine to arginine, leads to constitutive activation of an AGP1-lacZ reporter gene. An integrative plasmid carrying PTR3(Q439)-myc (pZL2323) was targeted to the PTR3 genomic locus in ptr3Δ cells carrying an AGP1-lacZ reporter gene (ZLY1917). The resultant transformant and wild-type control cells were grown in SD medium with or without leucine as indicated, and AGP1-lacZ expression was analyzed as described previously. (B) Ptr3(Q439R)-myc is hyperphosphorylated. Total cellular proteins from cells expressing the Ptr3(Q439R)-myc mutant or wild-type (WT) Ptr3-myc were separated by SDS-PAGE and probed with anti-myc antibody. The numbers (average from three independent experiments) indicate the ratios of the levels of hyperphosphorylated (hyperphos) to hypophosphorylated (hypophos) forms of Ptr3-myc.

  • FIG. 5.
    FIG. 5.

    An rts1Δ mutation results in the hyperphosphorylation of Ptr3 and the activation of SPS sensing. (A) An rts1Δ mutation constitutively activates AGP1-lacZ reporter gene expression. Wild-type (WT; ZLY044) and rts1Δ (ZLY2507) cells with an integrated AGP1-lacZ reporter gene were grown in SD medium with or without 0.02% leucine as indicated, and β-galactosidase activities were determined. (B) An rts1Δ mutation results in increased levels of hyperphosphorylated Ptr3. Wild-type (ZLY2142), ssy1Δ (ZLY2848), rts1Δ (ZLY2512), and ssy1Δ rts1Δ (ZLY2820) cells expressing Ptr3-myc from the genomic PTR3 locus were grown in SD medium with or without 0.02% leucine as indicated. Total cellular proteins were separated by SDS-PAGE and Ptr3-myc was probed with anti-myc antibody. The numbers indicate the ratios of the levels of hyperphosphorylated (hyperphos) and hypophosphorylated (hypophos) forms of Ptr3-myc. 3-Phosphoglycerate kinase (PGK) was used as a loading control. (C) An rts1Δ mutation does not affect expression of an HXT1-lacZ reporter gene. Wild-type (ZLY652) and rts1Δ (ZLY2538) cells with an integrated HXT1-lacZ reporter gene were grown in YNBCasRaffGal (− glucose) or YNBcasD (+ glucose) medium to mid-log phase, and β-galactosidase activities were determined. (D) An rts1Δ mutation partially rescues the cell growth defect in yck1Δ yck2ts mutant cells grown at a semipermissive temperature (34°C). Wild-type (LRB341), yck1Δ (ZLY2021), yck1Δ yck2ts (ZLY2518), and yck1Δ yck2tsrts1Δ (ZLY2627) cells were serially diluted fivefold and spotted onto YPD medium at either 23°C (permissive temperature) or 34°C and grown for 3 to 4 days.

  • FIG. 6.
    FIG. 6.

    Interaction of Ptr3 with itself or Ssy5 is not affected by the hyperphosphorylation of Ptr3. (A) Ssy5 is constitutively processed. ssy5Δ cells (ZLY1939) expressing nontagged Ssy5 (no tag; pZL736) or Ssy5-HA (pZL1668) as indicated were grown in SD medium with or without 0.02% leucine. Total cellular proteins were separated by SDS-PAGE, and Ssy5-HA was probed with anti-HA antibody. F and C indicate full-length Ssy5 and the C-terminal fragment of Ssy5, respectively. Alkaline phosphatase (ALP) was used as the loading control. (B) Ssy5 proteolytic processing is independent of Ssy1, Ptr3, or Grr1. Wild-type (WT; PLY126), ssy5Δ (RBY909), ssy1Δ ssy5Δ (RBY873), ptr3Δ ssy5Δ (RBY875), ssy1Δ ptr3Δ ssy5Δ (RBY951), and grr1Δ (ZLY175) cells transformed with a centromeric plasmid expressing Ssy5-HA (pZL1668) were grown in SD medium with 0.02% leucine to mid-log phase. HA-tagged proteins were detected as described for panel A. (C) Interaction between Ptr3 and Ssy5 is independent of Ptr3 hyperphosphorylation and of Ssy1. Indicated strains carrying centromeric plasmids expressing Ptr3-myc (pZL1239) and Ssy5-HA (pZL1668) were grown in SD medium with 0.02% leucine. Ptr3-myc was immunoprecipitated with anti-myc antibody. Ptr3-myc and Ssy5-HA were probed with anti-myc and anti-HA antibody, respectively. Ssy5 and Ssy5-C-HA indicate full-length and the C-terminal fragment of Ssy5. *, the heavy chain of anti-myc antibody. (D) Self-interaction of Ptr3 is independent of its phosphorylation status, Ssy1, and Ssy5. Indicated strains carrying centromeric plasmids expressing Ptr3-myc (pZL1239) and Ptr3-HA (pZL835) were grown in SD medium with 0.02% leucine. Ptr3-myc was immunoprecipitated with anti-myc antibody. Ptr3-myc and Ptr3-HA were probed with anti-myc and anti-HA antibodies, respectively.

  • FIG. 7.
    FIG. 7.

    The level of the N-terminal fragment of Ssy5 does not correlate with the activity of the SPS-sensing pathway. (A) Under steady-state conditions, a leucine-induced reduction of the N-terminal fragment of Ssy5, HA-Ssy5-N, is abolished in ssy1Δ but not in ptr3Δ single or ssy1Δ ptr3Δ double deletion mutant cells. ssy5Δ (RBY909), ssy1Δ ssy5Δ (RBY873), ptr3Δ ssy5Δ (RBY875), and ssy1Δ ptr3Δ ssy5Δ (RBY951) cells expressing an N-terminal 6× HA-tagged Ssy5 from a centromeric plasmid (pZL840) were grown in SD medium for at least six generations with or without 0.02% leucine as indicated. Total cellular proteins were separated by SDS-PAGE and HA-tagged proteins were probed with anti-HA antibody. Numbers at the bottom of the figure indicate the ratios of the levels of the N-terminal fragment of Ssy5, HA-Ssy5-N, to full-length HA-Ssy5. *, cross-reacting species. (B) Leucine-induced expression of an AGP1-lacZ reporter gene is abolished in ssy1Δ, ptr3Δ, and ssy1Δ ptr3Δ mutant cells. Wild-type cells (WT; RBY721) and ssy1Δ (HKY20), ptr3Δ (HKY31), and ssy1Δ ptr3Δ (RBY923) cells carrying an AGP1-lacZ report gene on a centromeric plasmid (pZL465) were grown in SD medium with or without 0.02% leucine as indicated for at least six generations to reach an optical density at 600 nm of 0.5 to 0.8, and β-galactosidase activities were determined.

  • FIG. 8.
    FIG. 8.

    Interactions between the N-terminal signal transduction domain of Ssy1, Ssy1N, and Ptr3, Yck1, and Ssy5. (A) A yeast two-hybrid analysis of protein interactions between Ssy1N and Ptr3, Ssy5, and Yck1. Yeast two-hybrid strains coexpressing GBD-Ssy1N or GBD-Lst8 and GAD-Lst8, Ptr3, Ssy5 or Yck1 were grown on medium as indicated. (B) A schematic representation of protein interactions in the amino acid-sensing pathway. Solid and dotted lines indicate protein interactions revealed by yeast two-hybrid analysis and coimmunoprecipitation, respectively.

  • FIG. 9.
    FIG. 9.

    A model for the regulation of the SPS-sensing pathway in yeast. Amino acid sensing is initiated by amino acid binding to Ssy1, which transmits signals through Ptr3 and Ssy5 and activates two transcription factors, Stp1 and Stp2, by enabling them to enter the nucleus. Once in the nucleus, Stp1/2 activate the expression of target genes encoding amino acid permeases (AAP). Ssy5, a protease, is constitutively processed into an N-terminal prodomain, Ssy5-N, and a C-terminal activity domain, Ssy5-C, which removes an N-terminal sequence from Stp1/2, allowing Stp1/2 to enter the nucleus. These two fragments of Ssy5 interact with each other after processing. Ptr3 constitutively interacts with Ssy5-C. In the absence of amino acids, Ptr3 is only partially phosphorylated (indicated by “P” in gray circles; “P” in black circles indicates robust phosphorylation). That form of Ptr3 is unable to activate the protease activity of Ssy5-C toward Stp1/2. Upon binding to amino acids, Ssy1 is likely to recruit Yck1/2, which is associated with the plasma membrane (PM) through prenylation (indicated by zigzag lines in PM), to hyperphosphorylate Ptr3. Hyperphosphorylated Ptr3 increases the Ssy5-C protease activity toward Stp1/2. The Rts1/PP2A phosphatase complex mediates SPS sensing by dephosphorylating Ptr3.

Tables

  • TABLE 1.

    Yeast strains used in this study

    StrainGenotypeSource/reference
    ZLY1917 MAT a ura3-52 ptr3-Δ15 AGP1::AGP1-lacZ::kanMX4This study
    HKY31 MAT a ura3-52 lys2-Δ201 ptr3-Δ15 17
    ZLY2142 MAT a ura3-52 ptr3-Δ15::PTR3-myc6::URA3 AGP1::AGP1-lacZ::kanMX4This study
    ZLY2916 MAT a ura3-52 ptr3-Δ15::PTR3-myc6::URA3 AGP1::AGP1-lacZ::kanMX4 grr1Δ::kanMX4This study
    RBY923 MAT a ura3-52 lys2-Δ201 ptr3-Δ15 ssy1::hisGThis study
    RBY875 MAT a ura3-52 lys2-Δ201 ptr3-Δ15 ssy5Δ::kanMX4This study
    RBY951 MAT a ura3-52 lys2-Δ201 ptr3-Δ15 ssy1::hisG ssy5Δ::kanMX4This study
    ZLY175 MAT a ura3-52 lys2-Δ201 grr1Δ::kanMX4This study
    RBY661 MAT a ura3 AGP1::AGP1-lacZ::kanMX4This study
    ZLY1915 MAT a ura3-52 ssy1-Δ13 AGP1::AGP1-lacZ::kanMX4This study
    LRB341 MAT a his3 leu2 ura3-52 25
    LRB264 MAT a his3 leu2 ura3-52 yck1-1::URA3 25
    ZLY2021 MAT a his3 leu2 ura3-52 yck1-1::ura3::kanMX4This study
    LRB343 MAT a his3 leu2 ura3-52 yck2::HIS3 25
    LRB362 MAT a his3 leu2 ura3-52 yck1-1::ura3 yck2-2ts 25
    ZLY044 MAT a ura3-52 AGP1::AGP1-lacZ::kanMX4This study
    ZLY2507 MAT a ura3-52 rts1Δ::kanMX4 AGP1::AGP1-lacZ::kanMX4This study
    ZLY2512 MAT a ura3-52 rts1Δ::kanMX4 ptr3-Δ15::PTR3-myc6::URA3 AGP1::AGP1-lacZ::kanMX4This study
    ZLY2848 MAT a ura3-52 ssy1-Δ13 ptr3-Δ15::PTR3-myc6::URA3 lys2-Δ201This study
    ZLY2820 MAT a ura3-52 ssy1-Δ13 rts1Δ::kanMX4 ptr3-Δ15::PTR3-myc6::URA3This study
    ZLY2535 MAT a ura3-52 rts1Δ::kanMX4This study
    ZLY2536 MAT a ura3-52 rts1Δ::kanMX4 ssy1-Δ 13This study
    ZLY652 MATα ura3 lys2 leu2 HXT1-lacZ::LEU2This study
    ZLY2538 MATα ura3 lys2 leu2 HXT1-lacZ::LEU2 rts1Δ::kanMX4This study
    ZLY2518 MAT a his3 leu2 ura3-52 yck1-1::ura3 yck2-2tsThis study
    ZLY2627 MAT a his3 leu2 ura3-52 yck1-1::ura3 yck2-2ts rts1Δ::kanMX4This study
    ZLY1939 MAT a ura3-52 ssy5Δ::kanMX4 AGP1::AGP1-lacZ::kanMX4This study
    RBY909 MAT a ura3-52 lys2-Δ 201 ssy5Δ::kanMX4This study
    RBY873 MAT a ura3-52 lys2-Δ201 ssy1-Δ13 ssy5Δ::kanMX4This study
    RBY721 MAT a ura3-52This study
    HKY20 MAT a ura3-52 lys2-Δ201 ssy1-Δ13 17
    S288c MAT a ura3 Butow lab stock
  • TABLE 2.

    Plasmids used in this study

    PlasmidDescriptionSource/reference
    pZL473pRS416 (CEN URA3) containing PTR3This study
    pZL1179pRS416 containing PTR3-myc; expresses Ptr3 with a 6× myc tag inserted between Ptr3 residues 157 and 158This study
    pEG-KG-YCK12 μm URA3 Leu2-d plasmid containing GAL1-GST-YCK1 36
    pEG-KG-YCK22 μm URA3 Leu2-d plasmid containing GAL1-GST-YCK2 36
    pZL1834pRS416 containing STP1-HA; expresses Stp1 with 3× HA tagged at the C-terminal endThis study
    pZL1949pRS416 containing HIS6-PTR3-myc; expresses Ptr3 with a six-His tag at the N-terminal end and a 9× myc tag inserted between residues 157 and 158 of Ptr3This study
    pZL2107pRS416 containing HIS6-PTR3(S321A)-mycThis study
    pZL2112pRS416 containing HIS6-PTR3(T635A)-mycThis study
    pZL2122pRS416 containing HIS6-PTR3(T525A)-mycThis study
    pZL2125pRS416 containing HIS6-PTR3(T525D)-mycThis study
    pZL2132pRS416 containing HIS6-PTR3(T525E)-mycThis study
    pZL2323Yip352 containing PTR3(Q439R)-myc
    pZL736pRS416 containing SSY5This study
    pZL1668pRS416 containing SSY5-HA; expresses Ssy5 with a 3× HA tag at the C-terminal endThis study
    pZL1239pRS417 (CEN LYS2) containing PTR3-myc; expresses Ptr3 with a 9× myc tag inserted between residues 157 and 158 of Ptr3This study
    pZL835pRS416 containing PTR3-HA; expresses Ptr3 with a 3× HA tag inserted between Ptr3 residues 157 and 158This study
    pZL840pRS416 containing HA-SSY5; expresses Ssy5 with a 6× HA tag at the N-terminal end of Ssy5aThis study
    pZL465pWCJ (CEN URA3) containing AGP1-lacZThis study
    pZL2230pRS417 containing STP1-HA; expresses Stp1p with 3× HA tagged at the C-terminal endThis study
    pGBKT7A yeast two-hybrid plasmid carrying Gal4 DNA binding domain encoding sequenceClontech Laboratories, Inc.
    pGBKT7-Lst8pGBKT7 containing the LST8 open reading frame (ORF)This study
    pGBKT7-SSY1NpGBKT7 carrying DNA sequence encoding amino acid residues 1-284 of Ssy1This study
    pACTIIA yeast two-hybrid plasmid carrying Gal4 DNA binding domain encoding sequenceClontech Laboratories, Inc.
    pACTII-LST8pACTII carrying the LST8 ORFThis study
    pACTII-PTR3pACTII carrying the PTR3 ORFThis study
    pACTII-SSY5pACTII carrying the SSY5 ORFThis study
    pACTII-YCK1pACTII carrying the YCK1 ORFThis study

    • ↵ a The primer pair gtcaACTAGTGTCACGGCGAATCGATCTAT and gtcaACGCGTCATCGGTATATCGAGTTTAC (lowercase letters indicate nucleotides introduced to facilitate digestion of PCR products by restriction endonucleases) was used to amplify a 1,003-bp DNA sequence containing the SSY5 promoter region plus the start codon by PCR. The primer pair gtcaACGCGTGTCAGATTTTTTGGTTTAAACAAGAAAAAGAACGA and gtcaGTCGACCTGTGAACCAAGGTACCTTC was used to amplify the SSY5 ORF plus a 376-bp 3′ untranslated region sequence. These two DNA fragments were digested with SpeI and MluI and with MluI and SalI, respectively, and cloned into SpeI- and SalI-digested pRS416 to form pZL737, thus introducing an MluI site right after the start codon of SSY5. Two tandem repeats of HA3 flanked by MluI sites were cloned into the MluI site of pZL737 to form pZL840.

Additional Files

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    Files in this Data Supplement:

    • Supplemental file 1 - Supplemental text; Fig. S1 (Mutations in five CKI consensus sites of PTR3 do not significantly affect AGP1-lacZ reporter gene expression), S2 (An rts1;delta mutation does not activate AGP1-lacZ expression in cocultured wild-type cells carrying an AGP1-lacZ reporter gene), S3 (The threonine 525-to-alanine mutation in Ptr3 does not affect its interaction with Ssy5-C), S4 (Mutations in SSY1 and PTR3 do not increase instability of Ssy5-C), and S5 (An N-terminal 6xHA-tagged Ssy5 is not constitutively active); and Tables S1 (Strains used) and S2 (Plasmids used)
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KEYWORDS

Amino Acids
Carrier Proteins
membrane proteins
Saccharomyces cerevisiae
Saccharomyces cerevisiae Proteins
signal transduction

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