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Activated Alleles of the Schizosaccharomyces pombegpa2+ Gα Gene Identify Residues Involved in GDP-GTP Exchange

F. Douglas Ivey, Francis X. Taglia, Fan Yang, Matthew M. Lander, David A. Kelly, Charles S. Hoffman
F. Douglas Ivey
Biology Department, Boston College, Chestnut Hill, Massachusetts 02467
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Francis X. Taglia
Biology Department, Boston College, Chestnut Hill, Massachusetts 02467
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Fan Yang
Biology Department, Boston College, Chestnut Hill, Massachusetts 02467
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Matthew M. Lander
Biology Department, Boston College, Chestnut Hill, Massachusetts 02467
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David A. Kelly
Biology Department, Boston College, Chestnut Hill, Massachusetts 02467
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Charles S. Hoffman
Biology Department, Boston College, Chestnut Hill, Massachusetts 02467
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  • For correspondence: hoffmacs@bc.edu
DOI: 10.1128/EC.00010-10
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  • Fig. 1.
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    Fig. 1.

    Coexpression of the Git5 Gβ enhances a two-hybrid interaction between the Git3 GPCR and the Gpa2 Gα subunit. (A) Strain YRG2 was transformed to Leu+ with a prey plasmid that expressed the carboxy-terminal half of Git3 beginning with the third cytoplasmic loop. This transformant was transformed to Trp+ G418r with bait and facilitating plasmids. The bait plasmids expressed either wild-type Gpa2+ or mutationally activated Gpa2(R176H). The facilitating plasmids expressed either the Git5 Gβ or the lambda cI repressor protein (−Git5 Gβ, as a negative control). Three independent transformants were replica plated onto SC-Leu, Trp, His with or without 7.5 mM 3AT and grown for 3 days before being photographed. (B) Growth of transformants on SC-Leu, Trp, His. (C) Growth of transformants on SC-Leu, Trp, His containing 7.5 mM 3AT. (D) X-Gal filter lift of transformants to detect β-galactosidase expression that resulted from the protein-protein interaction.

  • Fig. 2.
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    Fig. 2.

    Determination of the level of Gpa2 expression required to distinguish between wild-type and mutationally activated proteins. Strains CHP468 (gpa2Δ) and SLP29 (gpa2Δ git5Δ git11Δ) were transformed to Leu+ with pNMT41-1 (empty vector) (columns A), pDI2 (pNMT41-gpa2+) (columns B), pDI53 [pNMT41-gpa2(R176H)] (columns C), or pDI28 (pNMT41-myc-gpa2+) (columns D). Transformants were pregrown on PM-Leu medium with and without 10 μg/ml thiamine before being replica plated onto 5-FOA medium. The plates were incubated for 2 days at 30°C before being photographed.

  • Fig. 3.
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    Fig. 3.

    Crystal structure of transducin Gα bound to GDP, showing the locations of Gpa2 residues altered by activating mutations. The structure (Protein Data Base [PDB] ID 1TAG) was generated using RasMol. GDP is displayed in white using the spacefill format. (A and B) The 5 residues (T49 [blue], S149 [cyan], K270 [red], T325 [yellow], and V327 [green]) that contact the guanine nucleotide are displayed in the spacefill format. Two orientations of the structure are provided to show the contacts with GDP. (C) The 3 residues (I56 [blue], L57 [green], and F62 [red]) located in the linker 1 region are displayed in the spacefill format. (D) The 2 residues (S81 [blue] and V90 [green]) located in the αA helix of the helical domain are displayed in the spacefill format.

  • Fig. 4.
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    Fig. 4.

    Alignment of Gα subunits and S. pombe Rheb protein, together with identification of altered residues in activated S. pombe Gpa2 proteins. S. pombe (Sp) Gpa2 (CAB16244), S. cerevisiae (Sc) Gpa2 (NP_010937), human transducin α subunit (NP_000163), S. pombe Gpa1 (CAA21150; Gα of the pheromone pathway), and S. pombe Rhb1 (CAA22291) were aligned using Clustal W (44) and displayed using BOXSHADE 3.21. Identical residues are shaded in black. Conserved residues are shaded in gray. Dashes were introduced by the alignment software. The locations of altered residues that activate S. pombe Gpa2 (Table 3) are indicated above the alignment, while the locations of two altered residues that activate S. pombe Rhb1 (45) are indicated below the alignment.

Tables

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  • Table 1.

    Strain list

    StrainGenotype
    CHP468 h − ade6-M216 his7-32 ura4::fbp1-lacZ fbp1::ura4+gpa2Δ::ura4−
    SLP29 h − ade6-M216 his7-366 leu1-32 ura4::fbp1-lacZ fbp1::ura4+git5Δ::his7+git11Δ::kan gpa2Δ::ura4−
    DIP10 h − his7-366 leu1-32 ura4::fbp1-lacZ fbp1::ura4+git3Δ::kan gpa2Δ::ura4−
    MZP2 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2Δ::ura4+
    MZP5 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(R176H)
    MZP6 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(F62S)
    MZP7 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(K270E)
    MZP8 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(L57P)
    MZP9 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(K270N)
    MZP10 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(S149F)
    MZP12 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(T325A)
    MZP13 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(V90A)
    MZP14 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(V237A)
    MZP15 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(T49A)
    MZP18 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2+
    FYP1 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(I56S)
    FYP2 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(S81F)
    FYP3 h − his7-366 leu1-32 ura4::fbp1-lacZ git3Δ::kan gpa2(E184D)
  • Table 2.

    Analysis of DNA polymerases for frequency and diversity of mutations

    PolymeraseTotal no. of colonies screened% 5-FOAr% Confirmed by sequencingaNo. of transitions/totalNo. of transversions/total
    Pfu Turbo1,0001.6NDbNDND
    Taq 10,0006.51.68/80/8
    FailSafe1,05013.66.04/73/7
    • ↵a Eight of 32 sequenced Taq candidates contained ORF mutations. Seven of 16 sequenced FailSafe candidates contained ORF mutations.

    • ↵b ND, not determined.

  • Table 3.

    Bypass of git3Δ defect in glucose signaling by activated gpa2 allelesa

    gpa2 alleleDomainβ-Gal activity
    gpa2Δ1,240 ± 166
    gpa2 + 390 ± 101
    gpa2(I56S)Linker 115 ± 13
    gpa2(L57P)Linker 17 ± 1
    gpa2(F62S)Linker 19 ± 3
    gpa2(S81F)Helical13 ± 8
    gpa2(V90A)Helical8 ± 3
    gpa2(S149F)Helical7 ± 1
    gpa2(T49A)GTPase8 ± 1
    gpa2(R176H)GTPase5 ± 0
    gpa2(E184D)GTPase658 ± 90
    gpa2(K270E)GTPase7 ± 1
    gpa2(K270N)GTPase5 ± 3
    gpa2(T325A)GTPase37 ± 20
    gpa2(V327A)GTPase11 ± 4
    • ↵a β-Galactosidase activity was determined from three or four independent cultures grown overnight in yeast extract rich medium containing 8% glucose to a final density of ∼1 × 107 cells/ml. The assays were carried out as previously described (30). All strains carry a deletion of the git3+ GPCR gene, which can be suppressed by mutational activation of Gpa2 to restore repression of fbp1-lacZ expression. The values represent the mean specific activities per milligram of soluble protein ± standard errors.

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Activated Alleles of the Schizosaccharomyces pombegpa2+ Gα Gene Identify Residues Involved in GDP-GTP Exchange
F. Douglas Ivey, Francis X. Taglia, Fan Yang, Matthew M. Lander, David A. Kelly, Charles S. Hoffman
Eukaryotic Cell Apr 2010, 9 (4) 626-633; DOI: 10.1128/EC.00010-10

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Activated Alleles of the Schizosaccharomyces pombegpa2+ Gα Gene Identify Residues Involved in GDP-GTP Exchange
F. Douglas Ivey, Francis X. Taglia, Fan Yang, Matthew M. Lander, David A. Kelly, Charles S. Hoffman
Eukaryotic Cell Apr 2010, 9 (4) 626-633; DOI: 10.1128/EC.00010-10
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KEYWORDS

Alleles
GTP-Binding Protein alpha Subunits
Gene Expression Regulation, Fungal
Guanosine Diphosphate
Guanosine Triphosphate
Schizosaccharomyces
Schizosaccharomyces pombe Proteins

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