P-body formation was regulated by Ras/PKA signaling activity.
(A) Elevated signaling through the Ras/PKA pathway inhibited P-body formation in response to an acute glucose starvation. Strains expressing GFP-tagged versions of Dcp2, Dhh1, Pat1 and Xrn1 were transformed with either a vector control or a MET3-RAS2^val19 plasmid. Cells were grown to mid-log phase in SC-glucose medium and expression from the RAS2^val19 locus was induced by transferring cells to a medium lacking methionine for 2 hr. The cells were then starved for glucose for 15 min and visualized by fluorescence microscopy.
(B) The formation of P-bodies in early stationary phase was inhibited by elevated Ras/PKA signaling. Wild-type yeast with either a control vector or a MET3-RAS2^val19 plasmid were grown for 24 hr in an SC-glucose medium lacking methionine.
(C, D) The down-regulation of PKA activity was sufficient to induce P-body formation in glucose-replete conditions. In C, cells containing an analog-sensitive allele of TPK1, tpk1-as, were treated with 5 μM 1NM-PP1 for 15 min. In D, the cells were transformed with either a control vector or a high-copy PDE2 plasmid.

P-body formation was not influenced by either TORC1 or Snf1 signaling activity.
(A) Neither nitrogen deprivation or rapamycin treatment resulted in P-body formation. Strains expressing GFP-tagged versions of the indicated P-body markers were grown to mid-log phase in SC-glucose medium. The cells were then transferred to a medium lacking either a nitrogen source (-N) or glucose (-Glu), or treated with 200 ng/ml rapamycin (+Rap) for 15 min.
(B) The presence of rapamycin did not interfere with the P-body formation induced by glucose starvation. Cells were grown to mid-log phase in an SC-glucose medium and then transferred for 15 min to a medium lacking glucose that contained either 0 or 200 ng/ml rapamycin.
(C) Snf1 activity was not required for P-body formation in response to glucose starvation. Wild-type and snf1Δ cells were grown to mid-log phase in an SC-glucose medium and transferred to a medium lacking glucose (SC-D) for 15 min before fluorescence microscopy.

The Pat1 protein was a substrate for PKA.
(A) PKA phosphorylated Pat1 at both Ser-456 and Ser-457 in vitro. The indicated Pat1 variants were precipitated from cell extracts and either mock treated (-) or incubated with PKA and [γ-³²P] ATP. The reaction products were then separated on SDS-polyacrylamide gels and the level of PKA phosphorylation was assessed by autoradiography (³²P).
(B) Pat1 was phosphorylated by PKA in vivo. A GST-tagged Pat1 protein was precipitated and treated with λ phosphatase, as indicated. An aliquot of the beads from this latter reaction was washed and incubated with PKA and 2.5 mM ATP. The relative level of PKA phosphorylation was assessed by Western blotting with an anti-PKA substrate antibody (α-Sub), as described in the Experimental Procedures. PPase, λ phosphatase.
(C) Pat1 recognition by the anti-PKA substrate antibody required the presence of the two serine residues, Ser-456 and Ser-457. The indicated Pat1 variants were precipitated from cell extracts and the relative level of PKA phosphorylation was assessed by Western blotting with the anti-PKA substrate antibody.
(D) The in vivo level of the PKA phosphorylation on Pat1 was elevated in a strain over-expressing the PKA catalytic subunit, Tpk1.
(E) Recognition by the anti-PKA substrate antibody was lost following the inactivation of PKA. The tpk1-as strain was incubated with 5 μM 1NM-PP1 for 15 min to inactivate PKA.

The PKA-mediated inhibition of P-body formation was suppressed by the presence of a non-phosphorylatable form of Pat1.
(A) The PKA-dependent phosphorylation of Pat1 was rapidly lost upon glucose starvation. Cells expressing a myc-tagged Pat1 were grown to mid-log phase in an SC-glucose medium and then transferred to a medium lacking glucose (SC-D) for the indicated time. The level of PKA phosphorylation was assessed by Western blotting with the anti-PKA substrate antibody.
(B, C) The presence of the non-phosphorylatable variant, Pat1-AA, resulted in an elevated number of P-body foci in bcy1Δ cells. Wild-type and bcy1Δ cells expressing the indicated GFP-tagged Pat1 proteins were grown to mid-log phase and then transferred to a medium lacking glucose for 15 min. Representative fluorescence microscopy images are shown in B and the quantification of these data in C.
(D) The presence of the non-phosphorylatable Pat1 also suppressed the inhibitory effects of elevated PKA signaling on P-body formation during an extended glucose starvation (-Glu, 90 min) or growth into early stationary phase (1d).

The presence of the Pat1-EE variant disrupted Dhh1 recruitment to P-bodies.
(A) The Pat1-EE variant exhibited a weaker interaction with Dhh1 than the wild-type Pat1. The indicated myc-tagged Pat1 proteins were precipitated from yeast cell extracts and the relative level of the associated Dhh1-GFP was assessed by Western blotting.
(B) The presence of elevated Ras/PKA signaling activity resulted in less Pat1 binding to Dhh1. Wild-type cells expressing a myc-tagged Pat1 and Dhh1-GFP fusion protein were transformed with either a vector control (-) or RAS2^val19 plasmid (+). The myc-tagged Pat1 was immunoprecipitated from cell extracts and the relative level of the associated Dhh1-GFP was assessed by Western blotting.
(C) The formation of Dhh1 foci upon glucose starvation was impaired in strains containing the Pat1-EE variant. A pat1Δ strain expressing the GFP-tagged Dhh1 reporter and the indicated versions of Pat1 was grown to mid-log phase and transferred to a medium lacking glucose (SC-D) for 15 min.
(D) A histogram showing the fraction of cells in panel C that contained at least one large P-body focus following glucose starvation.

The PKA phosphorylation of Pat1 may influence stationary phase cell survival.
(A) The presence of the nonphosphorylatable Pat1-AA variant suppressed the loss of viability observed in stationary phase cultures of the RAS2^val19 mutant. pat1Δ cells containing either a control vector or a RAS2^val19 plasmid were grown for the indicated number of days in a YM-glucose minimal medium. The number of viable cells present was determined by plating out increasing dilutions of the cultures. The cells also contained a plasmid expressing either Pat1-SS or Pat1-AA, as indicated.
(B) Cells lacking Pat1 exhibited a decreased rate of survival in stationary phase. Wild-type or pat1Δ strains were grown for the indicated number of days and the number of viable cells in each culture was assessed.
(C) A model proposing that PKA activity specifically inhibits the aggregation step of P-body assembly. See text for further details.