Figure 1.
Loss of TPD3 Leads to a Decreased Serine/Threonine Phosphatase Activity and Altered Substrate Specificity of the PP2A C Subunit
The catalytic activity of anti-HA immunoprecipitates from lysates of wild-type (wt), tpd3Δ, rrd1Δ/rrd2Δ, and cdc55Δ/rts1Δ cells expressing HA-tagged PPH21 was analyzed by phosphatase assays towards (A) phosphorylase a (n = 12, mean ± standard deviation) or (B) para-nitrophenyl phosphate, pNPP (n = 4, mean ± standard deviation). An additional background measurement (corresponding to the wild-type strain containing the empty vector pYX142 was included for every assay (A and B) and subtracted from each measuring point (see Materials and Methods for more details).
Figure 2.
Decreased Catalytic Activity Is Intrinsic to the C Subunit
(A) Anti–HA-tag immunoprecipitates from lysates of wild-type (wt) cells containing an empty vector (control) or wild-type, tpd3Δ, rrd1Δ/rrd2Δ, cdc55Δ/rts1Δ, and ppm1Δ cells expressing HA-tagged PPH21 were analyzed by SDS-PAGE and immunoblotting. The blots were sequentially incubated with specific antibodies against HA-tag, RRD2, and TAP42. Lanes 4–6 were not adjacent on the original blot.
(B) Monomeric C subunits from the wild-type and the tpd3Δ strains were obtained by immunoprecipitation from lysates in which protein complexes had been disrupted by a basic pH shift, followed by neutralization (monomeric), or from control untreated lysates (complexed) (see Materials and Methods for details). The HA-PPH21 immunoprecipitates were analyzed by SDS-PAGE and immunoblotting.
(C) An aliquot of the monomeric HA-PPH21 immunoprecipitates was tested by phosphatase assays towards phosphorylase a (n = 3).
Figure 3.
RRD2 Interacts Physically and Functionally with TPD3
(A) HA-PPH21 or myc-RRD2 immunoprecipitates (IP) from lysates of wild-type (wt) cells co-expressing HA-PPH21 and myc-RRD2 were analyzed by SDS-PAGE and immunoblotting. The blots were sequentially incubated with anti-methyl PP2A/PPH21 (2A10), rabbit polyclonal anti-TPD3, anti-RRD2, anti-TAP42, and anti-PPH21 antibody. endog., endogenous.
(B) Anti–Myc-tag immunoprecipitates from lysates of wild-type cells co-expressing myc-RRD2 and HA-PPH21 were eluted by peptide competition and re-immunoprecipitated via HA-PPH21 (see Materials and Methods for details). “Control” indicates a wild-type strain containing both empty vectors. The re-immunoprecipitates were analyzed by SDS-PAGE and immunoblotting using specific antibodies.
(C) rrd1Δ/rrd2Δ and rrd1Δ/rrd2Δ/tpd3Δ cells containing the plasmid PYES2[myc-TPD3] were grown to log phase in complete dropout medium. Growth of the double and triple mutant was compared by spotting equal amounts of 10-fold serially diluted cells onto synthetic complete (SC) medium plates ± 1 g/l 5-fluoroorotic acid (5-FOA). The plates were incubated for 2 d (SC) or 5 d (SC + 5-FOA) at 30 °C. Screening on 5-FOA selects for the loss of the URA3 plasmid PYES2[myc-TPD3] and indicates dependency of viability on TPD3.
Figure 4.
Strains Impaired in C Subunit Biogenesis Display Decreased C Subunit Methylation and Increased PPE1 Binding
(A) Anti–HA-tag immunoprecipitates (IP) from lysates of wild-type (wt), tpd3Δ, rrd1Δ/rrd2Δ, and ppm1Δ cells expressing HA-tagged PPH21 were analyzed by SDS-PAGE and immunoblotting. C subunit methylation and the presence of PPE1 were analyzed using specific antibodies.
(B and C) HA-PPH21 immunoprecipitates from lysates of wild-type cells expressing HA-tagged PPH21 together with myc-tagged methylesterase (myc-PPE1) (overnight induction under the control of the GAL1 promoter), or containing an empty pYES2 vector (vector), were analyzed by SDS-PAGE/immunoblotting (B) and by phosphatase assays (C) towards phosphorylase a (n = 3). The blots were sequentially incubated with anti-methyl PP2A, anti-RRD2, rabbit polyclonal anti-TPD3, anti-PPE1, and anti-HA antibodies. endog., endogenous.
Lanes 4 and 5 (A) and lanes 2 and 3 (B) were not adjacent on the original blot.
Figure 5.
Deletion of PPE1 Restores C Subunit Catalytic Activity in the tpd3Δ, but Not in the rrd1Δ/rrd2Δ Strain
(A) HA-tagged PPH21 was immunoprecipitated from lysates of wild-type (wt), ppe1Δ, rrd1Δ/rrd2Δ, rrd1Δ/rrd2Δ/ppe1Δ, tpd3Δ, and tpd3Δ/ppe1Δ cells. The immunoprecipitates were separated by 10% SDS-PAGE and analyzed by immunoblotting with anti-HA, anti-methyl PP2A, monoclonal anti-TPD3, anti-CDC55, anti-RRD2, and anti-PPE1 antibodies.
(B) Aliquots of the anti–HA-tag immunoprecipitates used in (A) were assayed for phosphatase activity using phosphorylase a as substrate (n = 7).
(C) Logarithmically growing cultures of wild-type, ppe1Δ, rrd1Δ/rrd2Δ, rrd1Δ/rrd2Δ/ppe1Δ, tpd3Δ, and tpd3Δ/ppe1Δ were 10-fold serially diluted in YPD liquid medium, spotted on YPD plates, and incubated at 16 °C for 5 d, or at 30 °C or 37 °C for 3 d.
Figure 6.
C Subunit Methylation Is Required for the Restoration of C Subunit Activity in the tpd3Δppe1Δ Strain
HA-tagged PPH21 was immunoprecipitated from lysates of wild-type (wt), tpd3Δ, tpd3Δ/ppe1Δ, and tpd3Δ/ppe1Δ/ppm1Δ cells. The immunoprecipitates (IP) were (A) analyzed by 10% SDS-PAGE/immunoblotting and (B) assayed for phosphatase activity using phosphorylase a as substrate (n = 7).
Figure 7.
Model: RRD/PTPA-Dependent Generation of Active PP2A C Subunit Is Coupled to Holoenzyme Assembly and Regulated by Methyl-Esterase/Transferase Enzymes
Table 1.
Yeast Strains
Table 2.
Yeast Constructs