Cloning, characterization and subcellular localization of a gene encoding a human Ubiquitin-conjugating enzyme (E2) homologous to the Arabidopsis thaliana UBC-16 gene product.
Ubiquitin charging and activation of class III E2 enzymes has been directly linked to their nuclear import. It has not been published whether other classes E2s also abide by this mechanism. During the large-scale sequencing analysis of a human fetal brain cDNA library, we isolated a cDNA clone that is 2252 base pair in length, encoding a putative 162 amino acid protein, which shares high homology to Arabidopsis thaliana ubiquitin-conjugating enzyme 16 (Accession number NP_565110, 51% identity and 71% similarity) at protein level. Bioinformatics analysis revealed that the gene is composed of 7 exons, located on human chromosome 8q13-8q21.1, and that the predicted protein of the gene is a class I E2, for only composed of a conserved approximately 150-amino acid catalytic core, ubiquitin-conjugating enzyme E2 domain (UBC domain). In the C-terminal of the UBC domain sequence, there are two nuclear localization signals (NLSs). RT-PCR showed that this gene is ubiquitously expressed in 16 kinds of normal human tissues, but expression level is very low, unless in human heart, brain, liver, and pancreas. The subcellular localizations of the new human Ubiquitin conjugating enzyme E2 and its mutation were also examined, which showed that the nuclear localization of hUBC16 depended on two conditions: It has NLS, and at the same time, has enzyme active site, too, at least in HEK293 cells.
Yin
Gang
G
State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, P.R. China.
Ji
Chaoneng
C
Wu
Tong
T
Shen
Zhouliang
Z
Xu
Xin
X
Xie
Yi
Y
Mao
Yumin
Y
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D002874:Chromosome Mapping; D002875:Chromosomes / Q000737:chemistry; D003001:Cloning, Molecular; D019295:Computational Biology; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D005091:Exons; D015723:Gene Library; D017343:Genes, Plant; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D019913:Nuclear Localization Signals; D010940:Plant Proteins / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D025801:Ubiquitin / Q000737:chemistry; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics*
D000596:Amino Acids; D029681:Arabidopsis Proteins; D018076:DNA, Complementary; D019913:Nuclear Localization Signals; D010940:Plant Proteins; D012333:RNA, Messenger; D025801:Ubiquitin; C510625:UBC-16 protein, Arabidopsis; C510626:UBE2W protein, human; D044763:Ubiquitin-Conjugating Enzymes; D003545:Cysteine