Compared to other organism, the ABC protein family is significantly enlarged in plants with about 130 members in Arabidopsis thaliana and Oryza sativa. The ABC proteins are classified into eight subfamilies (ABCA – ABCG, ABCI) with ABCG subfamily being the largest and showing a reverse domain orientation (NBD N-terminal to the TMD) compared to the other subfamilies. In Arabidopsis, the ABCG subfamily comprises 28 half-size transporters (White Brown Complex (WBC)) and 15 plant- and fungi-specific Pleiotropic Drug Resistance (PDR) full-size transporters. Potential roles include heavy metal detoxification, pathogen response or the formation of physical barriers.
Based on knock-out studies, it is often difficult to conclusively demonstrate whether a single or various substrates are transported by a membrane protein. Furthermore, the new emerging role of ABC transporters in plants as importers requires clarification about the transport mechanisms. However, biochemical studies, helping to better understand the proteins and the transport process are rather limited. One reason is the necessity for purified protein in adequate quantities for biochemical and/or structural studies. Hence, it is imperative to have a suitable overexpression system that provides sufficient amounts of the protein in reasonable time. This is still a main issue concerning eukaryotic membrane proteins. Heterologous overexpression of membrane proteins is often challenging due to the size and hydrophobic nature of membrane proteins, toxicity of the protein to the host, misfolding, wrong localization or lack of the proper post-translational modification(s). Other studies also demonstrated that not only expression, but also cloning of membrane proteins, especially ABC transporters, may cause difficulties because the sequences are unstable in Escherichia coli. Despite these challenges, heterologous overexpression is for some subsequent applications indispensable. Members of the plant PDR family were expressed in eukaryotic systems, however, other studies also demonstrated that expression of PDRs is not always easily achieved. For example AtABCG37/PDR9 did not localize to the correct membranes when expressed in S. cerevisiae. It was subsequently expressed in HeLa cells and in Schizosaccharomyces pombe. Nicotiana plumbaginifolia PDR5 could neither be expressed in S. cerevisiae nor in S. pombe. Expression of NtPDR1 in the heterologous system S. cerevisiae was weak and unstable and the protein was not correctly localized. It was finally expressed in the homologous system. For N. plumbaginifolia PDR1, even cloning was not successful. We introduced the successful heterologous overexpression of AtABCG30/PDR2, AtABCG36/PDR8 and the half-size transporter AtABCG1/WBC1 using the P. pastoris expression system. The accomplishment of cloning and expression of large plant membrane proteins with high numbers of transmembrane helices is not always easily achieved. We evaluate and introduced the P. pastoris expression system for heterologous expression of plant ABC transporters belonging to the PDR subfamily and employed P. pastoris as a host for this protein family.
The majority of the analyzed half-size AtABCGs have been shown to be part of diffusion barrier formation. Diffusion barriers, such as cutin or suberin, which are deposited in the cell wall, enable plants to prevent water loss or serve as protective layers against many environmental stress factors, including drought and pathogen attack. Several half-size AtABCG transporters have been shown to be involved in pollen protection. Specifically, AtABCG26 has been shown to be important for nexine formation. A double mutant defective in AtABCG1 and AtABCG16 indicated a role in pollen nexine and intine layer formation, as well as in post-meiotic pollen development. Additionally, AtABCG9 acts together with the full-size transporter AtABCG31 in pollen coat maturation. In some cases, ABCG half-size transporters from the same phylogenetic clade act together in order to build up specific diffusion barriers. For example, AtABCG11, AtABCG12, and AtABCG13 are involved in cuticle formation. Another clade is formed by AtABCG1, AtABCG16, AtABCG2, AtABCG6, and AtABCG20. Amongst these, all clade members except for AtABCG1 and AtABCG16 were reported to be part of suberin formation in Arabidopsis roots and seeds. The suberin polymer consists of an aliphatic polyester, which is linked with phenolic components and associated with waxes. Typical monomers of the aliphatic suberin domain are w-hydroxy acids, a,w-dicarboxylic acids, fatty acids and alcohols. Here, glycerol is esterified to the w-hydroxy and a,w-dicarboxylic acids and the phenylpropanoid pathway derived phenolic compounds. The suberin monomers are synthesized in specific cell compartments and transported through the plasma membrane for final assembly into the suberin polymer in the apoplast. Current hypotheses for suberin precursor transport to the cell wall include transport via the secretory pathway, passive transport of oleophilic bodies, or transport by ABC transporters.
These data introduced for the first time, purification, characterization on enzymatic level, and direct functional assays of a P. pastoris expressed plant ABC transporter. Moreover, both approaches, biochemical assays as wells as root suberin analyses indicate the involvement of AtABCG1 in transport of aliphatic suberin precursors with higher chain length (C24 or C26) in Arabidopsis roots.
Only recently we started to analyze AKT1 and its interaction partners (CBL1, CBL2 and CIPK23) in vitro and obtain quantitative data on regulation and interaction.
Shanmugarajah, K., N. Linka, K. Grafe, S.H.J. Smits, A.P.M. Weber, J. Zeier & L. Schmitt, (2019) ABCG1 contributes to suberin formation in Arabidopsis thaliana roots. Sci Rep 9: 11381.
Grafe, K., K. Shanmugarajah, T. Zobel, S. Weidtkamp-Peters, D. Kleinschrodt, S.H.J. Smits & L. Schmitt, (2019) Cloning and expression of selected ABC transporters from the Arabidopsis thaliana ABCG family in Pichia pastoris. PLoS One 14: e0211156.