Transposon Mutagenesis Shows [URE3] Prion Pathology Prevented by a Ubiquitin-Targeting Protein: Evidence for Carbon/Nitrogen Assimilation Cross Talk and a Second Function for Ure2p in .

[URE3] is an amyloid-based prion of Ure2p, a regulator of nitrogen catabolism. While most "variants" of the [URE3] prion are toxic, mild variants that only slightly slow growth are more widely studied. The existence of several antiprion systems suggests that some components may be protecting cells from potential detrimental effects of mild [URE3] variants. Our extensive transposon mutagenesis showed that disruption of dramatically slows the growth of [URE3-1] strains. Ylr352wp is an F-box protein, directing selection of substrates for ubiquitination by a "cullin"-containing E ligase. For efficient ubiquitylation, cullin-dependent E ubiquitin ligases must be NEDDylated, modified by a ubiquitin-related peptide called NEDD8 (Rub1p in yeast). Indeed, we find that disruption of NEDDylation-related genes , , , and is also counterselected in our screen. We find that like Δ [URE3] strains, Δ Δ strains do not grow on nonfermentable carbon sources. Overexpression of Hap4p, a transcription factor stimulating expression of mitochondrial proteins, or mutation of , encoding glutamine synthetase, allows growth of ∆ [URE3] strains on glycerol media. Supplying proline as a nitrogen source shuts off the nitrogen catabolite repression (NCR) function of Ure2p, but does not slow growth of Δ strains, suggesting a distinct function of Ure2p in carbon catabolism. Also, mutations impair NCR, but actually relieve the growth defect of Δ [URE3] and Δ Δ strains, again showing that loss of NCR is not producing the growth defect and suggesting that Ure2p has another function. largely protects cells from the deleterious effects of otherwise mild [URE3] variants or of a mutation (the latter a rarer event), and we name it (lets [URE3]/ grow).