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Ricin A chain (RTA) undergoes retrograde trafficking and is postulated to use components of the endoplasmic reticulum (ER) associated degradation (ERAD) pathway to enter the cytosol to depurinate ribosomes. However, it is not known how RTA evades degradation by the proteasome after entry into the cytosol. We observed two distinct trafficking patterns among the precursor forms of wild type RTA and nontoxic variants tagged with enhanced green fluorescent protein (EGFP) at their C-termini in yeast. One group, which included wild type RTA, underwent ER-to-vacuole transport, while another group, which included the G83D variant, formed aggregates in the ER and was not transported to the vacuole. Peptide: N-glycanase (Png1), which catalyzes degradation of unfolded glycoproteins in the ERAD pathway affected depurination activity and toxicity of wild type RTA and G83D variant differently. PreG83D variant was deglycosylated by Png1 on the ER membrane, which reduced its depurination activity and toxicity by promoting its degradation. In contrast, wild type preRTA was deglycosylated by the free pool of Png1 in the cytosol, which increased its depurination activity, possibly by preventing its degradation. These results indicate that wild type RTA has a distinct requirement for Png1 compared to the G83D variant and is deglycosylated by Png1 in the cytosol as a possible strategy to avoid degradation by the ERAD pathway to reach the ribosome. [ABSTRACT FROM AUTHOR]