Project

Discipline

Lactococcus lactis; Transport mechanism; ABC transporter; Substrate identification; Plant

Lead
This project will contribute to better understand plant ABC transporters, especially to identify their substrates and reveal the detailed transport mechanism of ABC protein which will allow to place them in a physiological context.
Lay summary
ABC Proteine bilden eine der grössten Proteinfamilien und kommen in allen Organismen vor. Die Mehrzahl der ABC Proteine sind integrale Membranproteine und fungieren als ATP-abhängige Transporter die eine Vielzahl von Substraten wie Lipide, Pharmazeutika, Schwermetalle, Ionen und Hormone transportieren. Die Modelpflanze Arabidopsis enthält 129 ABC Proteine, aber das Substrat wurde bisher erst für 10 ABC Transporter eindeutig identifiziert. Für 20 weitere Gene wurde eine Funktion vorgeschlagen, aber die Substrate wurden bisher nicht eindeutig beschrieben und Transportdaten liegen für diese ABC Transporter auch nicht vor. Dieses Projekt hat das Ziel pflanzliche ABC Transporter besser zu verstehen, insbesondere dadurch, dass man den Transportern eindeutig Substrate zuordnen kann und den Transportmechanismus aufklärt. Das wird es erlauben, ihre physiologische Rolle besser zu verstehen. Um dieses Ziel zu erreichen werden wir das L. lactis System für pflanzliche ABC Transporter etablieren und damit ein Screening für Substrate durchführen.

Direct link to Lay Summary

Last update: 23.05.2017

The ABC protein family encompasses one of the largest protein families and is found in all phyla. The majority of ABC proteins are integral membrane proteins and act as ATP-driven transporters for a very wide range of substrates including lipids, drugs, heavy metals, ions and hormones. In the plant model-system Arabidopsis 129 are genes coding for ABC proteins. However so far the substrates have been identified only for about 10 ABC transporters. For about other 20 genes the function has been proposed, but the substrates have not been unequivocally identified and no transport data have been presented. I will establish a useful system to identify the substrates of ABC transporters starting with known ABC transporters using Lactococcus lactis subsp. and then I plan to identify substrates of unknown ABC transporters of Arabidopsis. L. lactis have a low intrinsic ATP hydrolytic activity while the hydrolytic activity of ABC transporters is strongly increased when they are exposed to their substrates. This will allow to identify new substrates for ABC transporters by adding a set of compounds and speed up the research on plant ABC transporters. Verification of the putatively transported substrates will be performed transport experiments with L. lactis plasma membrane vesicles and using UPLC-MS/MS. In addition so far no plant ABC transporter has been crystalized. The crystal structure of proteins is critical to understand their working mechanism. I will select the ABC transporters which show the highest expression in L. lactis and perform experiments to obtain amounts of protein suitable for structure generation. Through collaborations with specialists in protein structure and using the platform available at UZH, we will try to generate either 2-D or 3-D crystals that can be used for structure determination. To reach these goals, my project can be subdivided in four objectives, i) establish the L. lactis system for plant ABC transporters expression and substrate screening using a well-characterized ABC transporter; ii) apply the L. lactis system for investigating the substrate specificity of AtABCG14 which is implicated in cytokinine allocation; iii) select so far uncharacterized ABC transporters and identify their substrates using the L. lactis system; iv) investigate selected ABC transporter function using variable approaches: physiology, metabolomics, biochemistry, crystallization.All these subprojects have the aim to better understand ABC transporters, especially to identify their substrates and reveal the detailed transport mechanism of ABC protein which will allow to place them in a physiological context.