Abstract
Plant heterotrimeric G proteins are a major group of signaling molecules involved in regulation of critical processes including stress adaptation, seed size, grain quality and immune responses. Despite an abundance of in situ functional studies; purification of the individual subunits of the plant heterotrimer for biophysical and structural characterization and for studies on their interactions are lacking. In this study cloning of the genes encoding the β subunit AGB1 of A. thaliana and its γ-subunits AGG1 and AGG2 using different E. coli expression vectors and screening of expression in several strains are reported. AGB1 could be expressed albeit at very low levels and in all cases it was accompanied by overexpression of E. coli chaperone proteins. AGG1 could only be detected in inclusion body fractions, whereas AGG2 was obtained in soluble fractions and was purified. Purified AGB1 and AGG2 subunits were shown to dimerize in vitro. Further characterization of AGG2 by small angle X-ray scattering measurements and by dynamic light scattering revealed that AGG2 formed homodimers with extended shape in solution. These results are also consistent with those from circular dichroism spectroscopy which yielded 39.4% helical and 50% random coil content for AGG2. This is the first study showing heterologous expression of a plant heterotrimeric G protein β subunit individually and presenting its interaction with a plant γ-subunit in vitro. Results also show that the AGG2 subunit has a disordered structure, which would account for its role in diverse interactions for establishing selectivity in signal propagation.
Similar content being viewed by others
Data Availability
Data will be made available upon request.
References
Milligan G, Kostenis E (2009) Heterotrimeric G-proteins: a short history. Br J Pharmacol 147:S46–S55. https://doi.org/10.1038/sj.bjp.0706405
Pandey S, Vijayakumar A (2018) Emerging themes in heterotrimeric G-protein signaling in plants. Plant Sci 270:292–300. https://doi.org/10.1016/J.PLANTSCI.2018.03.001
Urano D, Chen J-G, Botella JR, Jones AM (2013) Heterotrimeric G protein signalling in the plant kingdom. Open Biol 3:120186. https://doi.org/10.1098/rsob.120186
Botella JR (2012) Can heterotrimeric G proteins help to feed the world? Trends Plant Sci 17:563–568. https://doi.org/10.1016/J.TPLANTS.2012.06.002
Ma H, Yanofsky MF, Meyerowitz EM (1990) Molecular cloning and characterization of GPA1, a G protein alpha subunit gene from Arabidopsis thaliana. Proc Natl Acad Sci 87:3821–3825. https://doi.org/10.1073/pnas.87.10.3821
Mason MG, Botella JR (2000) Completing the heterotrimer: Isolation and characterization of an Arabidopsis thaliana G protein gamma-subunit cDNA. Proc Natl Acad Sci 97:14784–14788. https://doi.org/10.1073/pnas.97.26.14784
Mason MG, Botella JR (2001) Isolation of a novel G-protein gamma-subunit from Arabidopsis thaliana and its interaction with Gbeta. Biochim Biophys Acta 1520:147–153. https://doi.org/10.1016/s0167-4781(01)00262-7
Chakravorty D, Trusov Y, Zhang W et al (2011) An atypical heterotrimeric G-protein γ-subunit is involved in guard cell K+-channel regulation and morphological development in Arabidopsis thaliana. Plant J 67:840–851. https://doi.org/10.1111/j.1365-313X.2011.04638.x
Jones JC, Temple BRS, Jones AM, Dohlman HG (2011) Functional reconstitution of an atypical G protein heterotrimer and regulator of G protein signaling protein (RGS1) from Arabidopsis thaliana. J Biol Chem 286:13143–13150. https://doi.org/10.1074/jbc.M110.190355
Maruta N, Trusov Y, Brenya E et al (2015) Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis. Plant Physiol 167:1004–1016. https://doi.org/10.1104/pp.114.255703
McCudden CR, Hains MD, Kimple RJ et al (2005) G-protein signaling: back to the future. Cell Mol Life Sci 62:551–577. https://doi.org/10.1007/s00018-004-4462-3
Howlett AC, Gray AJ, Hunter JM, Willardson BM (2009) Role of molecular chaperones in G protein beta5/regulator of G protein signaling dimer assembly and G protein betagamma dimer specificity. J Biol Chem 284:16386–16399. https://doi.org/10.1074/jbc.M900800200
Weiss CA, Garnaat CW, Mukai K et al (1994) Isolation of cDNAs encoding guanine nucleotide-binding protein beta-subunit homologues from maize (ZGB1) and Arabidopsis (AGB1). Proc Natl Acad Sci 91:9554–9558. https://doi.org/10.1073/pnas.91.20.9554
Trusov Y, Chakravorty D, Botella JR (2012) Diversity of heterotrimeric G-protein subunits in plants. BMC Res Notes. https://doi.org/10.1186/1756-0500-5-608
Xu Q, Zhao M, Wu K et al (2016) Emerging insights into heterotrimeric G protein signaling in plants. J Genet Genomics 43:495–502. https://doi.org/10.1016/j.jgg.2016.06.004
Roy Choudhury S, Li M, Lee V et al (2020) Flexible functional interactions between G-protein subunits contribute to the specificity of plant responses. Plant J. https://doi.org/10.1111/tpj.14714
Chen J-G, Ullah H, Temple B et al (2006) RACK1 mediates multiple hormone responsiveness and developmental processes in Arabidopsis. J Exp Bot 57:2697–2708. https://doi.org/10.1093/jxb/erl035
Wang S, Assmann SM, Fedoroff NV (2008) Characterization of the Arabidopsis heterotrimeric G protein. J Biol Chem 283:13913–13922. https://doi.org/10.1074/jbc.M801376200
Sambrook J, Russell DW (2001) “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory Press,3rd Edition
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Deleage G, Geourjon C (1993) An interactive graphic program for calculating the secondary structure content of proteins from circular dichroism spectrum. Comput Appl Biosci 9:197–199
Bohm G, Muhr R, Jaenicke R (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng 5:191–195
Konarev PV, Volkov VV, Sokolova AV et al (2003) PRIMUS: a windows PC-based system for small-angle scattering data analysis. J Appl Cryst 36:1277–1282
Svergun DI (1992) Determination of the regularization parameter in indirect-transform methods using perceptual criteria. J Appl Cryst 25:495–503
Franke D, Svergun DI (2009) DAMMIF, a program for rapid ab-initio shape determination in small-angle scattering. J Appl Crystallogr 42:342–346. https://doi.org/10.1107/S0021889809000338
Petoukhov MV, Konarev PV, Kikhney AG, Svergun DI (2007) ATSAS 2.1: towards automated and web-supported small-angle scattering data analysis. J Appl Crystallogr 40:s223–s228. https://doi.org/10.1107/S0021889807002853
Pandey S (2020) Plant receptor-like kinase signaling through heterotrimeric G-proteins. J Exp Bot 71:1742–1751. https://doi.org/10.1093/jxb/eraa016
Stateczny D, Oppenheimer J, Bommert P (2016) G protein signaling in plants: minus times minus equals plus. Curr Opin Plant Biol 34:127–135. https://doi.org/10.1016/j.pbi.2016.11.001
Bilecen K, Ozturk U, Duru A et al (2005) Triticum durum metallothionein. Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling. J Biol Chem. https://doi.org/10.1074/JBC.M412984200
Mogk A, Mayer MP, Deuerling E (2002) Mechanisms of protein folding: molecular chaperones and their application in biotechnology. ChemBioChem 3:807–814.
Kubota S, Kubota H, Nagata K (2006) Cytosolic chaperonin protects folding intermediates of Gbeta from aggregation by recognizing hydrophobic beta-strands. Proc Natl Acad Sci USA 103:8360–8365. https://doi.org/10.1073/pnas.0600195103
Robishaw JD, Kalman VK, Proulx KL (1992) Production, processing and partial purification of functional G protein βγ subunits in baculovirus-infected insect cells. Biochem J 286:677–680. https://doi.org/10.1042/bj2860677
Higgins JB, Casey PJ (1994) In vitro processing of recombinant G protein gamma subunits. Requirements for assembly of an active beta gamma complex. J Biol Chem 269:9067–9073
Gräslund S, Nordlund P, Weigelt J et al (2008) Protein production and purification. Nat Methods 5:135–146. https://doi.org/10.1038/nmeth.f.202
Parmar AS, Muschol M (2009) Hydration and hydrodynamic interactions of lysozyme: effects of chaotropic versus kosmotropic ions. Biophys J 97:590–598. https://doi.org/10.1016/j.bpj.2009.04.045
Sondek J, Bohm A, Lambright DG et al (1996) Crystal structure of a GA protein βγdimer at 2.1 Å resolution. Nature 379:369–374. https://doi.org/10.1038/379369a0
Wall MA, Coleman DE, Lee E et al (1995) The structure of the G protein heterotrimer Giα1β1γ2. Cell 83:1047–1058. https://doi.org/10.1016/0092-8674(95)90220-1
Morishita R, Kato K, Asano T (1994) A brain-specific gamma subunit of G protein freed from the corresponding beta subunit under non-denaturing conditions. FEBS Lett 337:23–26. https://doi.org/10.1016/0014-5793(94)80622-5
Morishita R, Ueda H, Kato K, Asano T (1998) Identification of two forms of the γ subunit of G protein, γ10 and γ11, in bovine lung and their tissue distribution in the rat. FEBS Lett 428:85–88. https://doi.org/10.1016/S0014-5793(98)00498-0
Yoshikawa DM, Hatwar M, Smrcka AV (2000) G protein β5 subunit interactions with α subunits and effectors. Biochemistry 39:11340–11347. https://doi.org/10.1021/BI0005557
Carson M, Johnson DH, McDonald H et al (2007) His-tag impact on structure. Acta Crystallogr D Biol Crystallogr 63:295–301. https://doi.org/10.1107/S0907444906052024
Acknowledgements
We would like to thank Dr. Dimitri Svergun and BIOSAXS group members at EMBL/DESY for their help in data collection and analysis and Dr. M. H. J. Koch for helpful discussions. This work was supported by the Sabanci University Internal Research Fund IACF08-00514 and by the bilateral program TUBITAK-Julich TBAG-U-155.
Funding
This work was supported by the Sabanci University Internal Research Fund IACF08-00514 and by the bilateral program TUBITAK-Julich TBAG-U-155
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
Authors declare no conflict of interest/competing interest.
Ethical Approval
Not applicable.
Informed Consent
Not applicable.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Kaplan-Türköz, B., Aktürk, A. & Sayers, Z. Oligomerization of A. thaliana Heterotrimeric G Protein Subunits AGB1 and AGG2 In Vitro. Protein J 39, 563–573 (2020). https://doi.org/10.1007/s10930-020-09914-z
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10930-020-09914-z