Based on genetic and affinity chromatography experiments, the karyopherin Kap123 was identified as an important player in the cellular response to the drug acadesine (AICAR, 5′-phosphoribosyl-5-amino-4-imidazole carboxamide). Since Kap123 mediates nuclear import of multiple proteins we hypothesized that AICAR could affect nuclear trafficking. The effect of acadesine (AICAR) on nuclear trafficking was assayed by comparing protein composition of enriched nuclear extracts from yeast treated or not with AICAR by label-free quantitative analysis. Total protein extracts from the same cultures were also assayed by quantitative mass spectrometry. A parallel analysis was performed on yeast cells grown in the presence of AICAR and overexpressing or not Kap123 to identify those proteins that are enriched in the nuclear fraction when Kap123 was overexpressed.