Prediction and Identification of Prox1 Nuclear Localization Signal in Chicken (Gallus gallus)
YUAN Chao, GAO Hong-Bo, ZHOU Lei, HAN Yi-Fan, DUAN Zhi-Qiang*
Key Laboratory of Animal Genetics,Breeding and Reproduction in Plateau Mountainous Region,Ministry of Education/Key Laboratory of Animal Genetics,Breeding and Reproduction of Guizhou Province/College of Animal Sciences,Guizhou University,Guiyang 550025,China
Abstract：Prox1 (prospero-related homeobox protein 1) is a transcription factor that plays an important role in embryonic development and carcinogenesis. This study aimed to predict and identify the nuclear localization signal (NLS) in chicken (Gallus gallus) Prox1 protein. Chicken Prox1 protein was truncated to determine the NLS region by fluorescence observation and subcellular localization analysis. The online software was then used to predict the putative NLS (pNLS) in Prox1 truncates,and the NLS was confirmed by examining the subcellular localization of the pNLS deletants. In addition,the conservation and nuclear import ability of chicken Prox1 NLS were analyzed. Moreover,the key basic amino acids determining the nuclear localization of Prox1 NLS were identified by point mutation test. The results showed that the N-terminus (1~220 aa) of chicken Prox1 protein had nuclear localization characteristics and two pNLS including pNLS1 (15KRRR18) and pNLS2 (163RAKRAR168) existed in this region by NLS software prediction. The subcellular localization analysis of pNLS deletants revealed that pNLS1 and pNLS2 codetermined the nuclear localization of chicken Prox1 protein. In addition,the NLS1 and NLS2 motifs in chicken Prox1 protein were conserved among other poultry,human (Homo sapiens) and different mammals,and had the same nuclear import ability as the NLS of SV40 large T antigen. Moreover,the results of basic amino acids mutation experiments showed that K15,R16,R18 and K165,R166,R168 were key amino acids in NLS1 and NLS2,respectively,which jointly determined nuclear localization of chicken Prox1 protein. This study confirmed two highly conserved NLSs present in chicken Prox1 protein,and both NLSs determined the nuclear localization together,which provided a reference for further studying molecular mechanism and function of the nuclear localization of chicken Prox1 protein.
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