Generation of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: evidence that ribosome depurination is not sufficient for cytotoxicity.
Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that depurinates the highly conserved alpha-sarcin/ricin loop in the large rRNA. Here, using site-directed mutagenesis and systematic deletion analysis from the 5' and the 3' ends of the PAP cDNA, we identified the amino acids important for ribosome depurination and cytotoxicity of PAP. Truncating the first 16 amino acids of PAP eliminated its cytotoxicity and the ability to depurinate ribosomes. Ribosome depurination gradually decreased upon the sequential deletion of C-terminal amino acids and was abolished when a stop codon was introduced at Glu-244. Cytotoxicity of the C-terminal deletion mutants was lost before their ability to depurinate ribosomes. Mutations in Tyr-123 at the active site affected cytotoxicity without altering the ribosome depurination ability. Total translation was not inhibited in yeast expressing the non-toxic Tyr-123 mutants, although ribosomes were depurinated. These mutants depurinated ribosomes only during their translation and could not depurinate ribosomes in trans in a translation-independent manner. A mutation in Leu-71 in the central domain affected cytotoxicity without altering the ability to depurinate ribosomes in trans and inhibit translation. These results demonstrate that the ability to depurinate ribosomes in trans in a catalytic manner is required for the inhibition of translation, but is not sufficient for cytotoxicity.
Hudak
Katalin A
KA
Biotechnology Center for Agriculture and the Environment and the Department of Plant Biology and Pathology, Cook College, Rutgers University, New Brunswick, NJ 08901-8520, USA.
Parikh
Bijal A
BA
Di
Rong
R
Baricevic
Marianne
M
Santana
Maria
M
Seskar
Mirjana
M
Tumer
Nilgun E
NE
D000225:Adenine / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D014176:Protein Biosynthesis / Q000187:drug effects; D017434:Protein Structure, Tertiary; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000737:chemistry / Q000187:drug effects*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D017384:Sequence Deletion; D014170:Transformation, Genetic
D010940:Plant Proteins; D012335:RNA, Ribosomal; D054789:Ribosome Inactivating Proteins, Type 1; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein; D000225:Adenine