Pokeweed antiviral protein (PAP) is a single-chain ribosome inactivating protein (RIP) that binds to ribosomes and depurinates the highly conserved alpha-sarcin/ricin loop (SRL) of the large subunit rRNA. Catalytic depurination of a specific adenine has been proposed to result in translation arrest and cytotoxicity. Here, we show that both precursor and mature forms of PAP are localized in the endoplasmic reticulum (ER) in yeast. The mature form is retro-translocated from the ER into the cytosol where it escapes degradation unlike the other substrates of the retro-translocation pathway. A mutation of a highly conserved asparagine residue at position 70 (N70A) delays ribosome depurination and the onset of translation arrest. The ribosomes are eventually depurinated, yet cytotoxicity and loss of viability are markedly absent. Analysis of the variant protein, N70A, does not reveal any decrease in the rate of synthesis, subcellular localization, or the rate of transport into the cytosol. N70A destabilizes its own mRNA, binds to cap, and blocks cap dependent translation, as previously reported for the wild-type PAP. However, it cannot depurinate ribosomes in a translation-independent manner. These results demonstrate that N70 near the active-site pocket is required for depurination of cytosolic ribosomes but not for cap binding or mRNA destabilization, indicating that the activity of PAP on capped RNA can be uncoupled from its activity on rRNA. These findings suggest that the altered active site of PAP might accommodate a narrower range of substrates, thus reducing ribotoxicity while maintaining potential therapeutic benefits.