Generation of Active Protein Phosphatase 2A Is Coupled to Holoenzyme Assembly
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).