Carboxymethylation of the PP2A catalytic subunit in Saccharomyces cerevisiae is required for efficient interaction with the B-type subunits CDC55p and RTS1p

Protein phosphatase 2A (PP2A) is an essential eukaryotic serine/threonine p hosphatase known to play important roles in cell cycle regulation. Associat ion of different B-type targeting subunits with the heterodimeric core (A/C ) enzyme is known to be an important mechanism of regulating PP2A activity, substrate specificity, and localization. However, how the binding of these targeting subunits to the A/C heterodimer might be regulated is unknown. W e have used the budding yeast Saccharomyces cerevisiae as a model system to investigate the hypothesis that covalent modification of the C subunit (Pp h21p/Pph22p) carboxyl terminus modulates PP2A complex formation. Two approa ches were taken. First, S, cerevisiae cells were generated whose survival d epended on the expression of different carboxyl-terminal Pph21p mutants. Se cond, the major S, cerevisiae methyltransferase (Ppm1p) that catalyzes the methylation of the PH)2A C subunit carboxyl-terminal leucine was identified , and cells deleted for this methyltransferase were utilized for our studie s. Our results demonstrate that binding of the yeast B subunit, Cdc55p, to Pph21p was disrupted by either acidic substitution of potential carboxyl-te rminal phosphorylation sites on Pph21p or by deletion of the gene for Ppm1p , Loss of Cdc55p association was accompanied in each case by a large reduct ion in binding of the yeast A subunit, Tpd3p, to Pph21p, Moreover, decrease d Cdc55p and Tpd3p binding invariably resulted in nocodazole sensitivity, a known phenotype of CDC55 or TPD3 deletion. Furthermore, loss of methylatio n also greatly reduced the association of another yeast B-type subunit, Rts 1p, Thus, methylation of Pph21p is important for formation of PPSA trimeric and dimeric complexes, and consequently, for PP2A function. Taken together , our results indicate that methylation and phosphorylation may be mechanis ms by which the cell dynamically regulates PP2A complex formation and funct ion.