EGFP tags affect cellular localization of ATP7B mutants.

Min Zhu, Wang Ni, Yi Dong, Zhi-Ying Wu

CNS neuroscience & therapeutics 2013 May

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Wilson's disease is an autosomal recessive disorder of copper metabolism due to mutations within ATP7B gene. Clinical investigations indicate that ATP7B truncations are associated with an early age of onset when compared to its missense mutations. In vitro studies show that mislocalization of ATP7B mutants is involved in disease-causing mechanisms. Enhanced green fluorescent protein (EGFP) tags are commonly used in in vitro studies of cellular localization of ATP7B mutants. However, there is still much unknown about cellular localization of ATP7B truncations. Here, we subcloned full-length human wild-type, a missense mutation (T935M), and four truncating mutants (E332X, Q511X, Q547X, Q819X) of ATP7B into pEGFP-C1, pEGFP-N2 and pCMV-myc, and transfected Chinese hamster ovary (CHO) and SH-SY5Y cells with them, respectively. ATP7B truncations all showed a diffuse and homogenous distribution pattern within the cytosol of CHO and SH-SY5Y cells, whereas its wild-type proteins and T935M mutation were clustered in the Golgi apparatus. Furthermore, we found that EGFP tags at N- or C-terminal would severely affect cellular localization of ATP7B truncations, and EGFP tags at N-terminal also have an influence on T935M localization. EGFP tags may not be suitable for the detection of cellular localization of ATP7B mutants. © 2013 Blackwell Publishing Ltd.

Citation

Min Zhu, Wang Ni, Yi Dong, Zhi-Ying Wu. EGFP tags affect cellular localization of ATP7B mutants. CNS neuroscience & therapeutics. 2013 May;19(5):346-51