NAT10, a nucleolar acetyltransferase, participates in a variety of cellular processes including ribosome biogenesis and DNA damage response. Immunohistochemistry staining showed that cytoplasmic and membranous NAT10 is related to the clinical pathologic characteristics in human cancer tissues. However, the mechanism about how NAT10 translocates from the nucleolus to cytoplasm and membrane is unclear. Here, we obtain a NAT10 deletion mutant localizing in cytoplasm and membrane. Bioinformatics analysis showed that residues 68-75 and 989-1018 are two potential nuclear localization signals (NLS) of NAT10. GFP-NAT10 deletion mutant (Delta 989-1018) predominantly translocates into cytoplasm with faint signal retained in the nucleolus, while GFP-NAT10(Delta 68-75) still remains in the nucleolus and nucleoplasm, indicating residues 989-1018 is the main nucleolar localization signal (NuLS). GFP-NAT10-D3, with both fragments (residues 68-75 and 989-1018) deleted, completely excludes from the nucleolus and translocates to cytoplasm and membrane. Therefore, complete NuLSs of NAT10 should include residues 68-75 and 989-1018. The cytoplasmic and membranous NAT10 mutant (Flag-NAT10-D3) colocalizes with alpha-tubulin in cytoplasm and with integrin on cell membrane. Importantly, Flag-NAT10-D3 promotes alpha-tubulin acetylation and stabilizes microtubules. Consequently, Flag-NAT10-D3 promotes migration and invasion in hepatocellular carcinoma (HCC) cells. Statistical analysis of immunohistochemistry staining of NAT10 in HCC tissues demonstrates that the cytoplasmic NAT10 is correlated with poorer prognosis compared with nuclear NAT10, while the membranous NAT10 predicts the poorest clinical outcome of the patients. We thus provide the evidence for the function of cytoplasmic and membranous NAT10 in the metastasis and prognosis of HCC patients. (C) 2018 Elsevier Inc. All rights reserved.