Protein phosphatase 2A (PP2A) is a prime example of the multisubunit architecture of protein serine/threonine phosphatases. Until substrate-specific PP2A holoenzymes assemble, a constitutively active, but nonspecific, catalytic C subunit would constitute a risk to the cell. While it has been assumed that the severe proliferation impairment of yeast lacking the structural PP2A subunit, TPD3, is due to the unrestricted activity of the C subunit, we recently obtained evidence for the existence of the C subunit in a low-activity conformation that requires the RRD/PTPA proteins for the switch into the active conformation. To study whether and how maturation of the C subunit is coupled with holoenzyme assembly, we analyzed PP2A biogenesis in yeast. Here we show that the generation of the catalytically active C subunit depends on the physical and functional interaction between RRD2 and the structural subunit, TPD3. The phenotype of the tpd3Δ strain is therefore caused by impaired, rather than increased, PP2A activity. TPD3/RRD2-dependent C subunit maturation is under the surveillance of the PP2A methylesterase, PPE1, which upon malfunction of PP2A biogenesis, prevents premature generation of the active C subunit and holoenzyme assembly by counteracting the untimely methylation of the C subunit. We propose a novel model of PP2A biogenesis in which a tightly controlled activation cascade protects cells from untargeted activity of the free catalytic PP2A subunit.
Multisubunit enzymes, such as protein phosphatase 2A, consist of a catalytic subunit and one of several regulatory subunits that are responsible for substrate specificity. Whereas this molecular architecture enables the assembly of a few components into many different substrate-specific enzymes, it possesses an inherent danger in the form of the uncomplexed catalytic subunit with its unspecific phosphatase activity. Until substrate-specific complexes assemble, the catalytic subunit would constitute a risk to the cell if no control mechanisms existed. We recently obtained evidence for the existence of the catalytic subunit in a low-activity conformation that requires an activator for the switch into the active conformation. This requirement suggested that the existing model of protein phosphatase 2A biogenesis was incomplete, because it could not explain how the activity of the catalytic subunit is kept in check until it is assembled with the substrate-targeting subunits. In this study, we provide evidence that the generation of the active catalytic subunit is coupled with and regulated by holoenzyme assembly. We propose a novel model of protein phosphatase biogenesis in which a tightly controlled activation cascade protects cells from the potential risk of unspecific dephosphorylation events.
Analysis of protein phosphatase 2A (PP2A) biogenesis in yeast suggests that a tightly controlled activation cascade, involving an interaction between the protein RRD2 and the structural subunit TPD3, protects cells from untargeted activity of the free catalytic PP2A subunit.