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Lipid flippases and vesicle budding: can bioimaging show the connection?
Rosa L. López-Marqués1, Lisbeth R. Poulsen1, katarina Meffert2, Thomas Günther-Pomorski1 and Michael
G. Palmgren1
1
Center for Membrane Pumps in Cells and Disease-PUMPKIN, Danish National Research Council, Faculty of Life
Sciences, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark
2
Humboldt- University Berlin, Faculty of Mathematics and Natural Science I, Institute of Biology, Invalidenstrasse 42,
10115 Berlin, Germany
The secretory pathway is involved in several vital cellular processes, including host-pathogen interactions,
nutrient and gravity sensing, and protein sorting [1-4]. Many elements of the secretory machinery in animals
and plants are still lacking or are poorly characterized [5-6]. In the past years, more and more evidence is
accumulating suggesting the involvement of a subfamily of P-type ATPases in vesicle formation. P-type
ATPases comprise a large family of membrane proteins with 8-12 transmembrane spans, involved in
pumping different physiologically-relevant substrates across biological membranes [7]. The members of the
P4 subfamily (also known as flippases) have been proposed to catalyze the energy-driven translocation of
lipids necessary for establishing transbilayer lipid asymmetry [8], a feature that seems to be necessary for
correct functioning of the cells [9,10]. Although direct evidence of the involvement of the P4-ATPases in
flipping natural lipids is still lacking, several of these proteins have been shown to catalyze the translocation
of fluorescent analogues of lipids [11,12]. Deletion of one or more P4-ATPase genes causes defects in vesicle
budding in various organisms [11-13] and some members of the yeast family have been shown to interact
with the vesiculation machinery [14,15].
In Arabidopsis, 12 members of the P4-ATPase family have been described, named ALA1-ALA12, for
Aminophospholipid ATPase [7]. To-date only two members (ALA1 and ALA3) have been partially
characterized. ALA1 is capable of transporting fluorescent analogues of phosphatidylserine (PS) when
expressed in yeast and its lack is directly related to the chilling sensitivity of an ALA1 antisense Arabidopsis
mutant [16]. ALA3 translocates preferentially analogues of phosphatidylethanolamine (PE) and an
Arabidopsis knock-out mutant lacking this protein shows a severe defect in vesicle production in specialized
root tip cells [12].
Currently we are characterizing another member of the ALA family, ALA2, capable of complementing yeast
mutants lacking the endogenous P4-ATPases. Using transient expression in tobacco epidermal cells followed
by confocal laser scanning microscopy, GFP:ALA2 was localized to highly mobile vesicular structures (1-4
µm in diameter). As many as 40 vesicles per cell could be detected. Our recent work suggests that these
vesicular structures are derived from the pre-vacuolar compartment as a result of ALA2 activity. At the
moment we are trying to use our tobacco expression system in combination with other techniques to prove
the direct involvement of ALA2 in the formation of these vesicular structures.
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