Published online 10 August 2004
Nucleic Acids Research, Vol. 32 No. 14 © Oxford University Press 2004; all rights reserved
Generation of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: evidence that ribosome depurination is not sufficient for cytotoxicity
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
* To whom correspondence should be addressed. Tel: +1 732 932 8165; Fax: +1 732 932 6535; Email: tumer{at}aesop.rutgers.edu
Present addresses: K. Hudak, Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada
M. Santana, Centro de Biotecnologia, Instituto de Estudios Avanzados Universidad Simon Bolivar (IDEA-USB), Apartado 17606, Caracas 1025A, Venezuela
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Received April 7, 2004; Revised June 24, 2004; Accepted July 21, 2004
Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that depurinates the highly conserved 
-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.
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