Secretory pathway pumps – University of Copenhagen

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Plant and Environmental Sciences > Research > Transport Biology > Secretory pathway pumps

Secretory pathway pumps

The interest of the group is to understand the mechanisms underlying lipid translocation across biological membranes and vesicle formation along the secretory pathway. Our recent data suggests that these two processes are connected by the action of a family of protein pumps known as P4-ATPases (also called flippases).

P4-ATPases belong to a family of cation-transporting proteins, the P-type ATPases, involved in relevant physiological functions, such as the generation of electrochemical gradients across membranes or cell detoxification.

We currently focus on three parallel research lines: 1) How do P4-ATPases translocate lipids across membranes? 2) How is lipid translocation regulated in living cells? 3) What is the physiological relevance of plant P4-ATPases?

To unravel these questions we use a broad range of techniques based on homologous and heterologous expression systems: transient expression in tobacco epidermal cells, bioimaging, molecular cloning, mutant generation and characterization, analysis of protein-protein interactions, membrane isolation and protein purification, activity assays, functional complementation studies, plant phenotypical analysis.

Selected figures from our publication in Plant Cell (2008) showing different aspects in which the flippase group is interested. From left to right: i) GUS-staining of an Arabidopsis root tip showing expression of the flippase ALA3; ii) electron-micrograph of Arabidopsis root cells specialized in secretion; iii) tobacco epidermal leaf cell expressing YFP-tagged ALA3; iv) proposed mechanism of action of flippases in lipid translocation.

 

The Arabidopsis flippase ALA2 localizes to the prevacuolar compartment. Right panel, fluorescence of ALA2 tagged with Green Fluorescent Protein (GFP). Middle panel, fluorescence of an endoplasmatic reticulum protein marker tagged with Yellow Fluorescent Protein (YFP). Left, overlay of both fluorescent images.

 

Selected publications:

  • Costa S, Marek M, Axelsen KB, Theorin L, Pomorski TG and López-Marqués RL, Role of posttranslational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase, Biochemical Journal (2016) in press, doi: 10.1042/BCJ20160207.

  • Poulsen LR, Palmgren MG and López-Marqués RL, Transient expression of P-type ATPases in tobacco epidermal cells in P-Type ATPases: Methods and Protocols, Maike Bublitz (Ed.), Methods in Molecular Biology, 1377 (2016) 383-393. doi: 10.1007/978-1-4939-3179-8_34.

  • Jensen MS, Costa S, Günther-Pomorski T and López-Marqués RL, Cell-based lipid flippase assay employing fluorescent lipid derivatives in P-Type ATPases: Methods and Protocols, Maike Bublitz (Ed.), Methods in Molecular Biology, 1377 (2016) 371-382. doi: 10.1007/978-1-4939-3179-8_33.

  • Poulsen LR+, López-Marqués RL+, Pedas PR, Mcdowell SC, Kunze R, Harper JF, Pomorski TG and Palmgren MG, A phospholipid uptake system in the model plant Arabidopsis thaliana. Nature Comms, 6 (2015), 7649. (+shared co-first author).

  • McDowell SC, López-Marqués RL, Cohen T, Brown E, Rosenberg A, Palmgren MG, Harper JF, Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 Impairs Pollen Fitness and Alters the Pollen Tube Plasma Membrane, Frontiers in Plant Science, section Plant Evolution and Development 6 (2015), doi 10.3389/fpls.2015.00197.

  • López-Marqués RL§, Poulsen LR, Bailly A, Geisler M, Pomorski TG, Palmgren MG. Structure and mechanism of ATP-dependent phospholipid transporters, Biochim Biophys Acta 1850 (2015), 461-475. (§ Corresponding author).