Functional characterization of the yeast Ppz1 phosphatase inhibitory subunit Hal3: a mutagenesis study.
Saccharomyces cerevisiae Hal3 is a conserved protein that binds the carboxyl-terminal catalytic domain of the PP1c (protein phosphatase 1)-related phosphatase Ppz1 and potently inhibits its activity, thus modulating all of the characterized functions so far of the phosphatase. It is unknown how Hal3 binds to Ppz1 and inhibits its activity. Although it contains a putative protein phosphatase 1c binding-like sequence (263KLHVLF268), mutagenesis analysis suggests that this motif is not required for Ppz1 binding and inhibition. The mutation of the conserved His378 (possibly involved in dehydrogenase catalytic activity) did not impair Hal3 functions or Ppz1 binding. Random mutagenesis of the 228 residue-conserved central region of Hal3 followed by a loss-of-function screen allowed the identification of nine residues important for Ppz1-related Hal3 functions. Seven of these residues cluster in a relatively small region spanning from amino acid 446 to 480. Several mutations affected Ppz1 binding and inhibition in vitro, whereas changes in Glu460 and Val462 did not alter binding but resulted in Hal3 versions unable to inhibit the phosphatase. Therefore, there are independent Hal3 structural elements required for Ppz1 binding and inhibition. S. cerevisiae encodes a protein (Vhs3) structurally related to Hal3. Recent evidence suggests that both mutations are synthetically lethal. Surprisingly, versions of Hal3 carrying mutations that strongly affected Ppz1 binding or inhibitory capacity were able to complement lethality. In contrast, the mutation of His378 did not. This finding suggests that Hal3 may have both Ppz1-dependent and independent functions involving different structural elements.
Muñoz
Iván
I
Department de Bioquímica i Biología Molecular, Universitat Autónoma de Barcelona, Cerdanyola 08193, Barcelona, Spain.
Ruiz
Amparo
A
Marquina
Maribel
M
Barceló
Anna
A
Albert
Armando
A
Ariño
Joaquín
J
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002384:Catalysis; D018797:Cell Cycle Proteins / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry*; D005816:Genetic Complementation Test; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D010749:Phosphoprotein Phosphatases / Q000737:chemistry*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014633:Valine / Q000737:chemistry
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; C092936:SIS2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D018698:Glutamic Acid; D006639:Histidine; C075119:PPZ1 protein, S cerevisiae; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D010744:Phosphoric Monoester Hydrolases; D014633:Valine