[Translational frameshift may be occur in p11, an interaction protein of Cx31, in yeast].
Connexin 31 is a member of connexin family. The carboxy-terminal cytosolic domain of connexin 31 contains several potential phosphorylation sites. In this work, a yeast two-hybrid protein interaction screen have been used to identify proteins that bind to the carboxy-terminus of connexin 31, and the p11 protein, an unique member of S100 protein family, and one of the two subunits of the annexin II tetramer was isolated. Interestingly, from yeast two-hybrid AD's coding sequence, three different reading frames of p11 DNA sequence were found,which come from different AD plasmids. By constructing AD plasmids using p11 ORF or 5' UTR, the protein coding by p11 ORF bind to connexin 31, while polypeptides coding by three kinds of 5 UTR did not bind to connexin 31, suggesting a translational frameshift of p11 fusion protein. To construct baits by dividing connexin 31 C-terminus into two domain, the p11 binding domain of connexin 31 was found located between 206-237 codons. The plasmid Cx31CT-pGEX-4T-2 was constructed for expression and purification of GST-Cx31CT; and p11-pQE30 for expression and purification of 6xHis-p11. In vitro binding assay showed that recombinant Cx31 interacted with recombinant p11.
Huang
Liang-Qun
LQ
National Laboratory of Medical Genetics of China, Changsha 410078, China. nlmglcy@public.cs.hn.cn
Shi
Xiao-Liu
XL
Tan
Zhi-Ping
ZP
Fang
Hai-Yan
HY
Zheng
Duo
D
Xia
Jia-Hui
JH
Xia
Kun
K
D020121:5' Untranslated Regions / Q000235:genetics; D017306:Annexin A2; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D018965:Frameshifting, Ribosomal; D017930:Genes, Reporter / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D009418:S100 Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D020798:Two-Hybrid System Techniques; D000099248:S100 Calcium Binding Protein A10
D020121:5' Untranslated Regions; D017306:Annexin A2; D002135:Calcium-Binding Proteins; D017630:Connexins; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D009418:S100 Proteins; D000099248:S100 Calcium Binding Protein A10; C068044:GJB3 protein, human