In vitro analysis of metabolite transport proteins

Marc Sven Roell, Franziska Kuhnert, Shirin Zamani-Nour, Andreas P.M. Weber

Research output: Chapter in Book/Conference paperChapterpeer-review

1 Citation (Scopus)

Abstract

The photorespiratory cycle is distributed over four cellular compartments, the chloroplast, peroxisomes, cytoplasm, and mitochondria. Shuttling of photorespiratory intermediates between these compartments is essential to maintain the function of photorespiration. Specific transport proteins mediate the transport across biological membranes and represent important components of the cellular metabolism. Although significant progress was made in the last years on identifying and characterizing new transport proteins, the overall picture of intracellular metabolite transporters is still rather incomplete. The photorespiratory cycle requires at least 25 transmembrane transport steps; however to date only plastidic glycolate/glycerate transporter and the accessory 2-oxoglutarate/malate and glutamate/malate transporters as well as the mitochondrial transporter BOU1 have been identified. The characterization of transport proteins and defining their substrates and kinetics are still major challenges. Here we present a detailed set of protocols for the in vitro characterization of transport proteins. We provide protocols for the isolation of recombinant transport protein expressed in E. coli or Saccharomyces cerevisiae and the extraction of total leaf membrane protein for in vitro analysis of transporter proteins. Further we explain the process of reconstituting transport proteins in artificial lipid vesicles and elucidate the details of transport assays.

Original languageEnglish
Title of host publicationPhotorespiration
Subtitle of host publicationMethods and Protocols
EditorsAlisdair R. Fernie, Hermann Bauwe, Andreas P.M. Weber
Place of PublicationUSA
PublisherHumana Press Inc.
Pages83-96
Number of pages14
ISBN (Electronic)978-1-4939-7225-8
ISBN (Print)978-1-4939-8422-0, 978-1-4939-7224-1
DOIs
Publication statusPublished - 2017
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume1653
ISSN (Print)1064-3745

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