Functional expression of human PP2Ac in yeast permits the identification of novel C-terminal and dominant-negative mutant forms.
The protein phosphatase 2A (PP2A) holoenzyme is structurally conserved among eukaryotes. This reflects a conservation of function in vivo because the human catalytic subunit (PP2Ac) functionally replaced the endogenous PP2Ac of Saccharomyces cerevisiae and bound the yeast regulatory PR65/A subunit (Tpd3p) forming a dimer. Yeast was employed as a novel system for mutagenesis and functional analysis of human PP2Ac, revealing that the invariant C-terminal leucine residue, a site of regulatory methylation, is apparently dispensable for protein function. However, truncated forms of human PP2Ac lacking larger portions of the C terminus exerted a dominant interfering effect, as did several mutant forms containing a substitution mutation. Computer modeling of PP2Ac structure revealed that interfering amino acid substitutions clustered to the active site, and consistently, the PP2Ac-L199P mutant protein was catalytically impaired despite binding Tpd3p. Thus, interfering forms of PP2Ac titrate regulatory subunits and/or substrates into non-productive complexes and will serve as useful tools for studying PP2A function in mammalian cells. The transgenic approach employed here, involving a simple screen for interfering mutants, may be applicable generally to the analysis of structure-function relationships within protein phosphatases and other conserved proteins and demonstrates further the utility of yeast for analyzing gene function.
Evans
D R
DR
Friedrich Miescher Institute, Maulbeerstrasse 66, CH-4058 Basel, Switzerland.
Myles
T
T
Hofsteenge
J
J
Hemmings
B A
BA
D000483:Alleles; D020134:Catalytic Domain; D003198:Computer Simulation; D006801:Humans; D007930:Leucine; D009154:Mutation; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000502:physiology; D054648:Protein Phosphatase 2; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013329:Structure-Activity Relationship
D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D007930:Leucine