Far3 and five interacting proteins prevent premature recovery from pheromone arrest in the budding yeast Saccharomyces cerevisiae - PubMed (original) (raw)

Far3 and five interacting proteins prevent premature recovery from pheromone arrest in the budding yeast Saccharomyces cerevisiae

Hilary A Kemp et al. Mol Cell Biol. 2003 Mar.

Abstract

In budding yeast, diffusible mating pheromones initiate a signaling pathway that culminates in several responses, including cell cycle arrest. Only a handful of genes required for the interface between pheromone response and the cell cycle have been identified, among them FAR1 and FAR3; of these, only FAR1 has been extensively characterized. In an effort to learn about the mechanism by which Far3 acts, we used the two-hybrid method to identify interacting proteins. We identified five previously uncharacterized open reading frames, dubbed FAR7, FAR8, FAR9, FAR10, and FAR11, that cause a far3-like pheromone arrest defect when disrupted. Using two-hybrid and coimmunoprecipitation analysis, we found that all six Far proteins interact with each other. Moreover, velocity sedimentation experiments suggest that Far3 and Far7 to Far11 form a complex. The phenotype of a sextuple far3far7-far11 mutant is no more severe than any single mutant. Thus, FAR3 and FAR7 to FAR11 all participate in the same pathway leading to G1 arrest. These mutants initially arrest in response to pheromone but resume budding after 10 h. Under these conditions, wild-type cells fail to resume budding even after several days whereas far1 mutant cells resume budding within 1 h. We conclude that the FAR3-dependent arrest pathway is functionally distinct from that which employs FAR1.

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Figures

FIG. 1.

FIG. 1.

Pheromone response pathway. Aspects of pheromone signaling relevant to this work are depicted. Although not indicated in this diagram, the α and γ subunits of the heterotrimeric G protein are tethered to the plasma membrane. MAPK, mitogen-activated protein kinase.

FIG. 2.

FIG. 2.

Characterization of far7 to far11 null mutants by halo assay. Halo assays were performed as described in Materials and Methods, using 2 μg of α-factor applied to a lawn of MATa cells. Strains were as follows: wild type (WT), SY2227; far1, SY2673; far3, SY4062; far7, SY4059; far8, SY4061; far9, SY4060; far10, SY4063; and far11, SY4064.

FIG. 3.

FIG. 3.

Far3 and Far7 to Far11 protein structure and homology. (A) Far3 and Far7 to Far11 protein domains. Regions predicted to encode CC, FHA, and TM domains are indicated by grey, red, and white boxes, respectively. Residues spanning each domain are indicated above the box in smaller type. (B) Far11 homologs in eukaryotes. A ∼700-amino-acid region of similarity was identified in Sc/Far11 to contain between 15 and 27% identity to the indicated homologous sequences from other organisms; in Sc/Far11, this region is indicated by grey shading. In protein sequences from other organisms, the region of homology, identified by iterative BLAST, is also indicated by grey shading. Domains 1 and 2, each containing absolutely conserved amino acid residues, are indicated by pink and blue shading, respectively. Predicted TM domains are indicated by white boxes. For panels A and B, the number of residues in the protein product is indicated in bold type. (C) Alignment of domain 1, containing four absolutely conserved amino acid residues over 33-amino-acid stretch. (D) Alignment of domain 2, containing 14 absolutely conserved residues over ∼250 amino acids. For panels C and D, red capital letters in the consensus indicate absolutely conserved residues and blue indicates weakly conserved amino acids. ! is either I or V; # represents any of NDQE; $ is L or M; % is either F or Y. For panels B to D, proteins of known function are as follows: Sc, S. cerevisiae Far11; Nc, N. crassa HAM-2. Hypothetical gene products in other eukaryotes are as follows: Sp, S. pombe SPBC27B12.04c; Hs1, H. sapiens FLJ14743; Hs2, H. sapiens KIAA1170; Mf, M. fascicularis hypothetical product of the AB050515 locus; Ag, A. gambiae agCP10939; Dm, D. melanogaster CG11526; Ce, C. elegans F10E7.8. For all panels, sequence analysis was performed as described in Materials and Methods.

FIG. 4.

FIG. 4.

Two-hybrid interactions among Far3 and Far7 to Far9. Two-hybrid tests were performed as described in Materials and Methods. Fusion proteins were tested in pairwise combinations consisting of a bait (Gal4BD fused to the N terminus of full-length Far3 or Far7) and a target (Gal4AD alone or Gal4AD fused to the N terminus of a Far protein or protein fragment). BD-Far3 and BD-Far7 baits were expressed from pSL2785 and pSL2786, respectively. Target proteins were expressed from plasmids as follows: AD alone from pGAD-C1; AD-Far7A (AD plus the C terminus of Far7, amino acids 65 to 222) from pSL2789; AD-Far8 (AD fused to full-length Far8) from pSL2790; AD-Far9B (AD plus the internal fragment of Far9, amino acids 138 to 572) from pSL2792. Interactions were assayed by patching independent isolates in duplicate to SD-Trp Leu and then replica plating to SD-Trp Leu His and SD-Trp Leu Ade. Growth on both tester media indicated a positive result. Patch tests were repeated at least four times using different isolates each time; the results of a typical patch test are shown.

FIG. 5.

FIG. 5.

Coprecipitation of Far3 with Far7 to Far11. Strains containing myc-tagged Far7 to Far11 were subjected to IP with a polyclonal rabbit HA antibody. Proteins contained in the IP were separated on duplicate SDS-PAGE gels and analyzed by Western blotting using monoclonal mouse antibodies to detect Far3-HA and myc-tagged Far7 to Far11. Pre-IP lysates were also analyzed by Western blotting to detect myc-tagged Far proteins or the loading control, Dpm1. Each myc-tagged Far protein was tested in isogenic strains with the following alterations: far3 mutant plus empty vector (YEp352), far3 mutant plus high-copy-number Far3-HA (pSL2784), and wild-type plus plasmid-borne HA tag (pSL2771). Far10-myc and Far11-myc were also tested in a far3 far9 background that contained the high-copy-number Far3-HA plasmid (pSL2784). Lane letters indicate strain details and experimental conditions, as follows: A, far3 〈YEp352〉 + IP α-HA (polyclonal rabbit anti-HA antibody); B, FAR3 〈pSL2771〉 + IP α-HA; C, far3 〈pSL2784〉 no IP α-HA; D, far3 〈pSL2784〉 + IP α-HA; E, far3 far9 〈pSL2784〉 + IP α-HA; X, 1:2 dilution of the concentrations in panel D. Strains used in each lane were as follows: Far7-myc (A, SY4065; B, SY4078; C and D, SY4066); Far8-myc (A, SY4067; B, SY4079; C and D, SY4068); Far9-myc (A, SY4069; B, SY4080; C and D, SY4070); Far10-myc (A, SY4071; B, SY4081; C and D, SY4072; E, SY4077); Far11-myc (A, SY4073; C, D, and X, SY4074; E, SY4084).

FIG. 6.

FIG. 6.

Physical interactions among Far3 and Far7 to Far11. Solid lines represent two-hybrid interactions. Dashed lines indicate interactions found by coIP. Black lines denote interactions discovered in the course of this study. Green lines denote interactions, either two-hybrid (32, 62) or coIP (21, 27), found by other groups.

FIG. 7.

FIG. 7.

Sucrose velocity gradients of Far3 and Far7 to Far11. (A) Cosedimentation of the Far proteins on sucrose velocity gradients. Lysates from strains containing epitope-tagged Far3 and Far7 to Far11 were separated on 15 to 40% continuous sucrose velocity gradients. Fractions were collected from the top and analyzed by Western blotting to detect each of the tagged Far proteins. Markers of known size were also separated on sucrose gradients. The fraction in which a particular size marker peaked is indicated below the corresponding lane. (B) Coprecipitation of Far3 with Far7 to Far11 in IPs from the sucrose gradient fraction peak. Far3-HA was immunoprecipitated from sucrose gradient fractions in which it was the most abundant; the presence of Far proteins in the IP was detected by Western blotting as described in Materials and Methods. Pregradient lysates and the gradient fractions used for IP were also analyzed by Western blotting to detect epitope-tagged Far proteins or the loading control, Dpm1. Lane letters indicate strain details and experimental conditions, as follows: B, FAR3 〈pSL2771〉 + IP α-HA; C, far3 〈pSL2784〉 no IP α-HA; D, far3 〈pSL2784〉 + IP α-HA. For both panels A and B, antibodies and strains are the same as those used in the original coIP experiments (Fig. 5). The asterisk (∗) next to the Far11-myc lanes in both panels A and B indicates that the 130-kDa cleavage product is shown, as opposed to the 160-kDa full-length protein.

FIG. 8.

FIG. 8.

Pheromone response in single and multiple mutants of far3 and far7 to far11. (A) Growth on pheromone. Serial dilutions of wild-type (WT) and mutant strains were spotted onto YEPD alone and YEPD + 0.1 μg of α-factor per ml. Growth was assessed after 3 days at 30°C. The experiment was repeated more than 20 times with the same results. The results of a typical experiment are shown. (B) Signal transduction. Wild-type and mutant strains containing a genomically integrated FUS1::lacZ reporter were tested for basal (no α-factor) and induced (plus α-factor) levels of β-galactosidase, as described in Materials and Methods. Each time the assay was performed, four independent isolates of each strain were grown to log phase and then either assayed directly or induced with α-factor before the β-galactosidase levels were measured. The assay was repeated three times, with similar results. The results of a typical experiment is shown. For all experiments, strains were as follows: WT, SY2227; ste12, SY4085; far1, SY2673; far3, SY4062; far7, SY4059; far8, SY4061; far9, SY4060; far10, SY4063; far11, SY4064; sextuple far3 far7 to far11, SY4075.

FIG. 9.

FIG. 9.

Kinetics of far3 and far1 recovery from cell cycle arrest during pheromone exposure. Strains were grown on YEPD with or without α-factor, and the percentage of budded cells was determined over time as described in Materials and Methods. Briefly, 30 or more unbudded cells from each strain were dissected onto the appropriate agar plate and budding was determined by microscopic examination. Budded cells scored at 25 h consisted of microcolonies containing two or more cells. Cells which had not resumed budding by the 25-h time point never did, suggesting that they were irreversibly arrested or damaged by the isolation protocol. Visible colony formation was confirmed no later than 32 h from the zero time point. The experiment was repeated three times with similar results; the results of a typical trial are shown. Strains were as follows: wild type (WT), SY2227; far1, SY2673; far3, SY4062; sextuple far3 far7 to far11, SY4075.

FIG. 10.

FIG. 10.

Far3 and Far7 to Far11 with all two-hybrid interactors of known function. Two-hybrid interactions were identified in one or more published large-scale screens (32, 62). Protein function and localization (13) are indicated as follows. Filled yellow circles indicate nuclearly localized proteins. Proteins with an RNA-related function (transcription, RNA processing, nuclear export, or translation) are indicated by blue borders. Proteins with a DNA-related function (chromosome segregation; silencing; telomere length) are indicated by orange borders. Essential proteins (13) are denoted by red type.

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