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. 2008 Mar;20(3):658-76.
doi: 10.1105/tpc.107.054767. Epub 2008 Mar 14.

The Arabidopsis P4-ATPase ALA3 localizes to the golgi and requires a beta-subunit to function in lipid translocation and secretory vesicle formation

Lisbeth Rosager Poulsen  1 , Rosa Laura López-Marqués, Stephen C McDowell, Juha Okkeri, Dirk Licht, Alexander Schulz, Thomas Pomorski, Jeffrey F Harper, Michael Gjedde Palmgren
Affiliations

The Arabidopsis P4-ATPase ALA3 localizes to the golgi and requires a beta-subunit to function in lipid translocation and secretory vesicle formation

Lisbeth Rosager Poulsen et al. Plant Cell. 2008 Mar.

Abstract

Vesicle budding in eukaryotes depends on the activity of lipid translocases (P(4)-ATPases) that have been implicated in generating lipid asymmetry between the two leaflets of the membrane and in inducing membrane curvature. We show that Aminophospholipid ATPase3 (ALA3), a member of the P(4)-ATPase subfamily in Arabidopsis thaliana, localizes to the Golgi apparatus and that mutations of ALA3 result in impaired growth of roots and shoots. The growth defect is accompanied by failure of the root cap to release border cells involved in the secretion of molecules required for efficient root interaction with the environment, and ala3 mutants are devoid of the characteristic trans-Golgi proliferation of slime vesicles containing polysaccharides and enzymes for secretion. In yeast complementation experiments, ALA3 function requires interaction with members of a novel family of plant membrane-bound proteins, ALIS1 to ALIS5 (for ALA-Interacting Subunit), and in this host ALA3 and ALIS1 show strong affinity for each other. In planta, ALIS1, like ALA3, localizes to Golgi-like structures and is expressed in root peripheral columella cells. We propose that the ALIS1 protein is a beta-subunit of ALA3 and that this protein complex forms an important part of the Golgi machinery required for secretory processes during plant development.

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Figures

Figure 1.
Figure 1.
ala3 Mutants Are Deficient in Root and Shoot Growth. (A) Genomic organization of ALA3 showing the position of the T-DNA insertions in ala3-1 and ala3-4. Exons are represented by black boxes. LB, left border; nt, nucleotides. (B) Left border sequences of the T-DNA insertion. Lowercase, T-DNA sequence; uppercase, genomic sequence. (C) Representative example of the vegetative phenotype under short-day conditions of wild-type, ala3-1, ala3-4, and transgenic ala3-1 expressing GFP:ALA3 (Rescued) plants. Bar = 1 cm. (D) Diagram of root growth (average ± sd) for the wild type (closed diamonds; n = 40), ala3-1 (open squares; n = 19), and ala3-4 (open triangles; n = 18). (E) Representative examples of root growth for wild-type, ala3-4, and transgenic ala3-1 expressing GFP:ALA3 (Comp) plants. Similar results were obtained for both ala3 lines.
Figure 2.
Figure 2.
ALA3 Is Expressed in a Variety of Cell Types in Roots and Shoots. ProALA3:GUS studies were performed on different tissues: flower (A), silique (B), vascular tissue in young leaf (C), stomatal guard cells (D), root vascular tissue ([E] and [F]), emerging side root (G), columella root cap initials ([H] and [I]), and all cells of the columella root cap ([J] and [K]). (L) shows a schematic drawing of the root tip: tan, lateral root cap; light blue, columella initials; darker and darkest blue, columella root cap cells. c, central columella cell; p, peripheral columella cell. Bars = 0.5 mm ([A] to [C]) and 50 μm ([D] to [K]).
Figure 3.
Figure 3.
Border-Like Cells Stay Associated with the Root Cap in ala3 Mutants. Semithin sections of 5-d-old wild-type (A), ala3-1 (B), and ala3-4 (C) roots were analyzed by light microscopy after staining with crystal violet. b, border-like cell; c, central columella cell; p, peripheral columella cell. Bar = 50 μm.
Figure 4.
Figure 4.
ala3 Mutants Show a Defect in Slime Vesicle Production and the Specialized Hypertrophied trans-Golgi Stacks in Root Tip Peripheral Columella Cells. (A) to (C) Transmission electron microscopic overview of peripheral columella cells from the wild type, ala3-1, and ala3-4, respectively. (D) to (F) Main cellular structures of the same cells from the wild type, ala3-1, and ala3-4, respectively. (G) to (I) Enlarged view of Golgi stacks in peripheral columella cells of the wild type, ala3-1, and ala3-4, respectively. Bars = 1 μm. G, Golgi; HG, hypertrophied Golgi; M, mitochondria; SV, slime vesicles. Arrows indicate vacuole-like structures.
Figure 5.
Figure 5.
ALA3 Is Localized in the Golgi Apparatus in Planta. Fusion proteins of ALA3 and GFP were expressed, either stably in the roots of transformed Arabidopsis ala3-1 plants ([A] to [D]) or transiently in tobacco epidermal cells together with the Golgi marker ST:YFP ([E] to [H]). (A) Diagram of the root tip. Cells in green are peripheral columella cells. (B) Transmitted light differential interference contrast image of a peripheral columella cell from a rescued ala3-1 plant transformed with the GFP:ALA3 construct. The same cell is seen in (C). (C) GFP signal in a peripheral columella cell from a rescued transgenic plant expressing GFP:ALA3. The signal is erratic and stronger in some places than in others, as expected for GFP expression from vesicular bodies and/or the Golgi. (D) A control transformed ala3-1 line expressing GFP only. The GFP signal is distributed evenly throughout the cell, as expected for localization in the cytosol and nucleus. (E) GFP fluorescence of tobacco epidermal cells coexpressing GFP:ALA3 and ST:YFP. (F) YFP signal from the same cells (ST:YFP). (G) Overlay on a bright-field image. (H) Intensity plots of YFP and GFP fluorescence of the profile sketched in the merged image (magenta line). The y axis represents the intensity measured in arbitrary units. Magenta/red, YFP; green, GFP. Arrows indicate the positions of intracellular Golgi bodies. Bars = 10 μm.
Figure 6.
Figure 6.
Identification of a Family of Putative Subunits to ALA3. (A) The triple yeast mutant Δdrs2 Δdnf1 Δdnf2 expressing ALA3 under the control of a galactose-inducible promoter. ALA3 cannot complement the cold-sensitive phenotype at 18°C, in contrast with plasmid-borne DRS2. Empty, vector control; GAL, galactose; GLU, glucose. (B) Arabidopsis ALIS proteins and yeast Cdc50p belong to a family of ubiquitous eukaryotic proteins in which plant ALIS proteins evidently cluster. Unrooted phylogenetic analysis of Cdc50p homologs from Saccharomyces cerevisiae (YEAST), Homo sapiens (HUMAN), Leishmania donovani (Ld), Leishmania major (LEIMA), Drosophila melanogaster (DROME), Caenorhabditis elegans (CAEEL), Oryza sativa (ORYZA), Medicago truncatula (MEDTR), and Arabidopsis thaliana (marked in boldface). Export Protein Analysis System accession numbers are given except for those for Ld Ros3 (Q0P0L8) and Arabidopsis ALIS1 to ALIS5 (Q9LTW0, Q67YS6, Q9SLK2, Q9SA35, and Q9SAK5, respectively). Bootstrap values are expressed in percentages and placed at nodes. (C) RT-PCR analysis of ALIS1 to ALIS5 expression in different tissues of Arabidopsis. (D) Alignment of ALIS protein sequences derived from cloned cDNAs with those of yeast Cdc50p and Lem3p. Transmembrane domains are underlined, and a conserved predicted N-glycosylation site is marked in boldface. Conserved residues are shaded gray.
Figure 7.
Figure 7.
ALIS1 Is Expressed in a Variety of Cell Types in Roots and Shoots. Examination of GUS expression directed by the ALIS1 promoter was performed on different tissues: flower (A), silique ([B] and [C]), leaf vascular tissue (D), stomatal guard cells (E), root vascular tissue ([F] and [G]), emerging side root (H), columella root cap initials ([I] and [J]), and all cells of the columella root cap ([K] and [L]). Bars = 0.5 mm ([A] to [D]) and 50 μm ([E] to [L]).
Figure 8.
Figure 8.
ALIS1 Is Localized in the Golgi Apparatus in Planta. Fusion proteins of ALIS1 and GFP were transiently expressed in tobacco epidermal cells in concert with a Golgi marker (ST:YFP). Arrows indicate the positions of intracellular Golgi bodies. (A) GFP fluorescence, signal of GFP:ALIS1. (B) YFP signal (ST:YFP). (C) Overlay on a bright-field image. (D) Intensity plots of YFP and GFP fluorescence of the profile sketched in the merged image (blue line). The y axis represents the intensity measured in arbitrary units. Magenta/red, YFP; green, GFP. Bar = 10 μm.
Figure 9.
Figure 9.
ALA3 in Combination with ALIS Genes Functionally Complement the Yeast drs2 Cold-Sensitive Phenotype. The triple yeast mutant Δdrs2 Δdnf1 Δdnf2 expresses ALA3 and ALIS genes, either alone or in combination, under the control of a galactose-inducible promoter. ala3, ala3D413D; Empty, yeast cells transformed with a control plasmid; GAL, galactose; GLU, glucose. (A) ALA3 expressed in combination with ALIS allows the growth of the cold-sensitive yeast strain at 18°C. ala3D413A in combination with ALIS1 does not complement the cold-sensitive strain at 18°C. (B) Protein blot analysis of total yeast membranes harboring different heterologous proteins.
Figure 10.
Figure 10.
Subcellular Localization of ALA3 in Yeast Is Independent of the Presence of Colocalizing ALIS1. Total membranes purified from yeast expressing HA-ALA3 (A), RGSH6-ALIS1 (B), HA-ALA3 and RGSH6-ALIS1 (C), and plasmid-borne DRS2 (D) were subjected to sucrose density gradient fractionation. (E) shows the differential centrifugation of yeast membranes containing RGSH6-ALIS1, HA-ALA3, or HA-ALA3 and RGSH6-ALIS1. The proteins present in each fraction were determined by protein blot analysis. Membrane marker proteins are as follows: Pma1p, PM; Dpm1p, ER; Sed5p, Golgi apparatus.
Figure 11.
Figure 11.
ALA3 and ALIS1 Interact Directly in Vivo. (A) Schematic drawing of the principle of the split-ubiquitin assay used. Interaction between two protein partners, each carrying halves of ubiquitin, leads to the formation of a full active ubiquitin, causing the degradation of the reporter protein Ura3p. If Ura3p is present, it converts 5-FOA to a toxic compound; if it is degraded, no toxic compound is formed. Thus, growth of the transformed yeast on plates containing 5-FOA indicates a positive protein–protein interaction. (B) Cells transformed with plasmid-borne fusions of the desired proteins to either the N or C terminus of ubiquitin. As a positive control, AHA2 and its well-known interacting partner 14-3-3 (Jahn et al., 1997) were used. As a negative control, the soluble transcription factor Ste14p was tested against AHA2 and ALA3. Additionally, AHA2 was tested against ALA3 and ALIS1.
Figure 12.
Figure 12.
ALA3 and ALIS1 Cosolubilize and Can Be Copurified. Total yeast membranes bearing HA-ALA3 and RGSH6-ALIS1 were solubilized at different concentrations of lysophosphatidylcholine, and the solubilized fractions were subjected to protein blot analysis. (A) Intensity of immunodetected bands in the supernatant after solubilization. (B) Intensity of each band compared with the intensity for ALIS1 at the same detergent concentration. Closed squares, ALA3; open squares, ALIS1; closed triangles, Pma1p; open circles, Sed5p. Values shown are averages ± sd (n = 4). (C) RGSH6-ALIS1 isolation by Ni-affinity chromatography from the detergent-solubilized fractions. Fractions collected during the affinity purification procedure were visualized by Coomassie blue staining. Bands corresponding to the molecular masses expected for ALIS1 and ALA3 are observed (arrowheads). (D) Protein blot analysis using antibodies against the HA (ALA3) and RGSH6 (ALIS1) tags added to the proteins. Sol., solubilized proteins; FT, flow-through; W1 to W7, washing steps; El., eluted fraction.
Figure 13.
Figure 13.
Coexpression of ALA3 and ALIS1 Complements the Lipid Uptake Defect of the Δdrs2 Δdnf1 Δdnf2 Yeast Mutant. Internalization of NBD phospholipids by Δdrs2 Δdnf1 Δdnf2 mutant cells transformed with a control vector (Empty) or plasmids expressing different protein combinations. Yeast cells, preincubated with (+) or without (−) latrunculin A (lat.A), were labeled with NBD-lipid and then washed and analyzed by flow cytometry. (A) Coexpression of ALA3 and ALIS1 resulted in a population of cells with increased NBD-lipid uptake (arrow). Representative histograms of NBD-PE–labeled cells are shown. (B) Accumulation of NBD lipids is shown as a percentage of fluorescence intensity relative to control Δdrs2 Δdnf1 Δdnf2 mutant cells (Empty). One hundred percent corresponds to 37 ± 8 and 35 ± 5 arbitrary units (NBD-PE), 84 ± 20 and 72 ± 23 arbitrary units (NBD-PS), 29 ± 1 and 26 ± 1 arbitrary units (NBD-PC), and 83 ± 37 and 52 ± 23 arbitrary units (NBD-SM) in the absence and presence of latrunculin A, respectively. Results for NBD-PS are averages ± se from at least three independent experiments; all other data represent averages ± range of two independent experiments. ala3, ala3D413A.
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