Characterizing G Protein Signaling Components In Arabidopsis Using TAPa-LP System

The G-proteins act as critical molecular switches in diverse signal transduction pathways in eukaryotes. Similarly, in plant, the G-proteins play regulatory roles in multiple developmental processes ranging from seed germination and early seedling development to root development and organ shape determination. The repertoire of G-protein signaling complex is much simpler than in metazoans. Specifically, Arabidopsis, has only one canonical G alpha, one G beta, and two G gamma subunits, only one Regulator of G-protein Signaling (RGS) protein and several proteins, including AtPIRIN1, PLD1, PD1, and THF1. Ga subunits contain two domains, a domain involved in binding and hydrolyzing GTP and a unique helical domain that buries the GTP in the core of the protein. We only have known about a preliminary of the spatial structure of G-proteins in metazoans, but know little about spatial structure of G-proteins in plant. So future studies are expected to identify the mechanisms by which G-proteins regulate phenotypic and developmental plasticity and to clarify of structure-function relationships in G-proteins or Ga. People urgently need some new methods of Arabidopsis protein complex isolation.Therefore, we describe the application of tandem affinity purification-linker peptide (TAP-LP) strategy to the study of Ga subunit in Arabidopsis.Rigaut described the tandem affinity purification (TAP) system in yeast, a number of studies have arisen in recent years demonstrating the applicability of such a system in many other different organisms, such as mammalian cells, insect cells, tobacco leaves et al., TAP tags sometimes not only affect bait protein function, but also can react to or bind with other proteins. It is a serious problem if the protein that you want to produce is TAPa fusion proteins. But we have obtained important information that regulatory domains are located in G-protein N-terminal region or C-terminal region because tag protein is due to interference with the function of N-terminal region or C-terminal region of bait protein.In order to take full advantage of this situation, we suggest TAP system construct two types:one is a linker sequence between a bait protein and TAPa, the other is no linker sequence between a bait protein and TAP tags. The design of linker or no linker may play an important part in the fusion protein. Linker peptide can independently fold protein domains in a fusion construct.We had to try a few candidate sequences (3-20aa long) with varying degrees of flexibility/rigidity by computer. Sequences made up of Gly and Ser. By varying the number of Gly-Ser pairs we can modulate the flexibility of the linker. Ultimately, we have designed a linker peptide, GGGGSGGGGGS, its nucleic acid sequence is GGAGGAGGAGGATCA GGAGGAGGAGGAGGATCA, then we have constructed5vectors composed of linker or no linker. Our results demonstrate that the TAPa-linker peptide (TAPa-LP) system sometimes can enhance the activity of TAP fusion proteins in Arabidopsis.The TAPa-LP system provides us with a new research method of Ga subunit in Arabidopsis. Subsequently, we forecasted three-dimensional structure of fusion proteins by’ SWISS-MODEL. TAPa-LP system revealed that G-proteins play a complex regulation role in the different stage in plant development.We found that GTP-Ga is a positive regulator in leaf, stem, flower and silique development whereas GTP-Ga is a negative regulator in primary root and anthocyanins synthesis in seed, while heterotrimeric complex is a positive regulator in primary root, stem, and silique development, whereas heterotrimeric complex is a negative regulator for cell proliferation in leaf blade’s width direction. That is to say, GTP-Gα act as4positive regulators and2negative regulators, and heterotrimeric complex act as3positive regulators and1negative regulators. GTP-Gα and heterotrimeric complex play reversal roles in the root and leaf development. We speculate that the ratio of GTP-Gα and Gaβγ impact on or strongly impact on the growth of plants. Lack of GTP-Gα and Gaβγ in gpa1-4and fpt do not obviously showed a significant difference in plant growth, suggesting that lack of positive and negative regulation of G protein does not affect the growth of plants, and G-proteins is not the primary regulation of plant growth in the major regulation components. gpt is the highest among all plant types in the T2plants because gpt with35S promoter-GPA1promote the ratio of GTP-Gα and Gaβγ.The size of qpt plant morphology appeared to show tall and strong in T1stage but qpt have mostly died and a few grew very weak at an early age in the T2stage, while the size of gpt plant morphology grew normal size in T1stage but gpt is the strongest and highest among all plant types in the T2plants in the early age, hormone level of T1seed is different from level of T2seed. So regulations of plant growth depend not only on the ratio of GTP-Ga and Gaβγ but also on the hormone level. Plant growth is regulated by the plant hormones gibberellins (GA), brassinosteroids (BR), abscisic acid (ABA) and auxin (Ueguchi-Tanaka et al.,2000; Ullah et al.,2002), but gpt and qpt appeared to show the greatest differences between T1and T2stage. We call this phenomenon as the elongation effects of hormone, because hormones in seed of transgenic plant come from parental generation. We speculated that a model of G-protein’s role mechanism perhaps is dependent on the hormone signal transduction pathway or/and affect hormone synthesis. However, in Arabidopsis, we expected to reveal more components of the heterotrimeric G-protein signal transduction pathways, and to identify the mechanisms by which G-proteins regulate phenotypic and developmental plasticity.Our studies showed that TAPa or linker-TAPa sometimes impaired bait protein function in G-protein-TAPa or G-protein-linker-TAPa fusion proteins, previous studies also reported this problem, such as FHY1and HFR1. It is a serious problem if the protein that you want to produce is TAPa fusion proteins. But we have obtained important information that regulatory domains are located in G-protein region because tag protein is due to interference with the function of bait protein. It is now known that Ga display two independent regions in the three dimensional structure. In terms of logic, regulatory domain is likely located in the C-terminal region of Ga when one, two or three of GT, GPT, QT and QPT play a role in terms of G-protein phenotypic, Such as A stem, I primary root, A primary root, A silique size, I seed color and A leaf.It is well known that N-terminal region of G-protein can bind the Gβγ dimmer, so we speculated that regulatory domains are liable to be in the C-terminal region of G-protein rather than in the N-terminal region. Of course, this model needs a lot of experiments to be further validated.Several known interactions involving Ga were confirmed, that is to say, TAPa-LP system revealed that several interactions of Arabidopsis G-proteins have one Gp and one Gγ1. Point out with great regret, we have no found many novel potential interactions.We conclude that TAPa-LP, in combination with MS, can be used as an effective method for the studies of the Arabidopsis G-proteins.When monochromatic light passes through a homogeneous absorbing medium, the absorbance is proportional to the growth of concentration and thickness of the medium, which is Lambert-Beer law. The shade selection of protein solution magnetized for certain time from different angles makes different absorbance, which does not meet the Lambert-Beer law. Accordingly, we derive that the absorbance A is not only proportional to the concentration and thickness of the medium, but also proportional to the light area Ss of certain direction. For the same protein solution, we can obtain the absorbance A of six directions, and thus get six SS, the relative ratio of which will inevitably reveal plentiful information of the protein shape. The conformation of protein can be easily drawn out by software (MATLAB7.0.1) developed by computer. We have drawn out the molecular shape of lysozyme and bovine serum albumin. In brief, we have developed Lambert-Beer law (A=K-C-b-Ss) and a new method of exploring protein spatial structure.To sum up, we have developed TAPa-LP purification strategy applied to Arabidopsis protein complex isolation. We have developed Lambert-Beer law (A=K·C·b·Ss) and a new method of exploring protein spatial structure.