Subcellular Localization And Membrane Topology Of Arabidopsis Vitamin K Epoxide Reductase

With the Arabidopsis genome and rice genome sequencing finished, the study of plant genome began. The attentions of biologist mainly focus on subcellular localizations, interactions and dynamic changes of proteins. The study of subcellular localizations of proteins is essential to the understanding of morphogenesis, growth and stress tolerance, and is also an important part of functional genomics. The topology research is helpful to find out the three-dimensional structure and functions of proteins. Vitamin K epoxide reductase (VKOR) is an ER membrane protein in human and mammalian. The studies of VKOR and its homologs focused on mammalian and prokaryote, while the research of VKOR homologs in plants is almost blank. Thus, we chose Arabidopsis VKOR as the target protein to study the subcellular localization and structure of VKOR homologs in higher plants. In present study, we predicted subcellular localization and topology of Arabidopsis VKOR, and then confirmed the results by transient expression of GFP Fusion in Arabidopsis protoplast and alkaline phosphatase fusion in E. coli. The results are as follows:(1) Prediction of subcellular localization and transit peptide of Arabidopsis VKOR. At4g35760 gene from Arabidopsis was found in NCBI. The putative target location and transit peptide of AtVKOR was predicted online with TargetP, PredSL, BaCello, SLPFA, SLP-Local and ChloroP programs, respectively. The prediction results indicated Arabidopsis VKOR is most likely located in chloroplast and the first 45 amino acids from N-terminal may be a chloroplast transit peptide.(2) Construction of expression vector fused with gfp. At4g35760 gene was cloned using specific primers from Arabidopsis cDNA library, and the open reading frame (ORF) of transit peptide of At4g35760 gene was clone into pJIT163-gfp vector yielding a plant transient expression vector pJIT163-TP-gfp.(3) Determination of subcellular localization of Arabidopsis VKOR by transient expression in protoplasts. Arabidopsis protoplasts were extracted and transformed with pJIT163-TP-gfp and pJIT163-gfp mediated by PEG. Then the protoplasts were incubated 12-16 hours at 23℃and visualized with confocal microscopy. TP-GFP showed a perfect co-localization of the green GFP fluorescence with the red chlorophyll signal, while the GFP-alone as a negative control displayed green fluorescence in cytoplasm. The results indicate that Arabidopsis VKOR is localized in chloroplast and there is a transit peptide of first 45 amino acids from N terminal in VKOR.(4) Prediction of topology of Arabidopsis VKOR. Accroding to the amino acids sequence of Arabidopsis VKOR, online programs including TMHMM, HMM-TM, TMPred, TOPCONS, SOSUI, PHILIUS, PolyPhobius, HMMTOP and THUMUP were applied for predicting the membrane topology of VKOR. The prediction results indicated that Arabidopsis VKOR contain two domains: transmembrane domain (AtVKOR) and thioredoxin-like domain (AtDsbA). And there are four or five transmembrane segments in AtVKOR domain.(5) Analysis of topology of Arabidopsis VKOR by alkaline phosphatase fusion. After fusing membrane proteins to alkaline phosphatase, the changes of alkaline phosphatase activities could indicate orientation of these proteins. Accroding to the prediction results of topology, the segments before predicted transmembrane helices, from N-terminal to different sites of transmembrane segments, were cloned using specific primers from the full length of Arabidopsis VKOR cDNA and introduced to the N-terminal of AP in pDHB7744, resulting to five AP fusion expression vectors. These vectors were transferred to E. coli and determind the alkaline phosphatase activities to study the topology of VKOR.(6) Determination of topology of Arabidopsis VKOR by improved alkaline phosphatase fusion. Due to the poor results of alkaline phosphatase activities, we used a method of improved alkaline phosphatase fusion, namely'sandwich'fusion, to study the topology of VKOR further. The remaining segments of VKOR were cloned and introduced to the C-terminal of AP to construct seven'sandwich'fusions. Each of the sandwich fusions was transformed into E. coli. These transformed cells were streaked on LB solid medium containing IPTG and 5-bromo-4-chloro-3-indolyl-phosphate (XP) and incubated for two days at 37℃. Then the AP activities of the transformed cells were determined. There were three fusions showing high activities while the other four fusions showed low activites. According to these results, we suggest a model of topology of VKOR: There are four TM helices and a possible partially inserted segment ahead in Arabidopsis VKOR while both the N terminus and C terminus are located in the periplasm/lumen. Moreover, all the conserved cysteines face to the periplasm/lumen.