Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.
Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.
Hur
Y
Y
Center for Agricultural Molecular Biology, Rutgers University, Cook College, New Brunswick, NJ 08903-0231, USA.
Hwang
D J
DJ
Zoubenko
O
O
Coetzer
C
C
Uckun
F M
FM
Tumer
N E
NE
D003001:Cloning, Molecular; D018076:DNA, Complementary; D009154:Mutation; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D010957:Plasmids; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D054789:Ribosome Inactivating Proteins, Type 1; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D018076:DNA, Complementary; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein