Nucleolar proteins such as Nop1/fibrillarin are degraded by nucleophagy in nutrient-starved conditions. However, whether and how excess nucleolar proteins are removed in normal conditions is unknown. Here we show that overexpressed nucleolar protein Nop1 is toxic and degraded in nutrient-rich conditions in budding yeast. The degradation was dependent on proteasomes. The CUE domain-containing protein Def1 mediated the degradation via the CUE domain and alleviated toxicity of Nop1 over-expression. Def1 was recruited to overexpressed Nop1 in the nucleolus. Ubiquitin mutants compromised this recruitment. This study revealed that Def1 is a novel factor for ubiquitin-dependent degradation of excess nucleolar proteins. (C) 2019 Elsevier Inc. All rights reserved.