Transport rates for ammonia and water in yeast cells expressing <i>At</i>TIP2;1, <i>Hs</i>AQP1, or mutants of <i>Hs</i>AQP1 mimicking the selectivity filter of <i>At</i>TIP2;1.

<p>Transport rates for ammonia and water in yeast cells expressing <i>At</i>TIP2;1, <i>Hs</i>AQP1, or mutants of <i>Hs</i>AQP1 mimicking the selectivity filter of <i>At</i>TIP2;1.</p

Functional assays.

<p>(A) Water permeability of liposomes with and without inserted <i>At</i>TIP2;1 measured at different pH values. Stopped-flow experiments with 100 mM hyperosmolar shift present high protein-facilitated conductivities at cytosolic and vacuolar pH. Relative single exponential rate constants of ca. 110 s⁻¹ at LPR 30 demonstrate the ability to sustain a highly water-permeable vacuole. (B) Water permeability of purified <i>At</i>TIP2;1 (LPR 50) is inhibited by mercury as previously shown in oocytes [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002411#pbio.1002411.ref021" target="_blank">21</a>]. (C) Ammonia uptake monitored by increased internal fluorescence after exchange of 20 mM NaCl with 20 mM NH₄Cl, corresponding to an initial NH₃ gradient of 4.5 μM. Proteoliposomes with <i>At</i>TIP2;1 (green) show higher permeability than equally-sized control liposomes (grey). Fitted curves yielding single exponential rates are also shown. (D) Summary of rates at different ammonia gradients. Equations and correlation coefficients are given for linear fitting of averaged single exponential rates as a function of total initial concentration gradient of NH₃/NH₄⁺. Error bars represent the standard deviation of the rate within a set of approximately ten stopped-flow recordings per liposome sample. The underlying data of panels A–D can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002411#pbio.1002411.s001" target="_blank">S1 Data</a>.</p

Growth complementation of ammonium uptake-defective yeast strain by mutants of <i>Hs</i>AQP1.

<p>The 31019b yeast strain (<i>Δmep1–3</i>) was transformed with the empty vector pYeDP60u or with pYeDP60u carrying cDNA encoding the positive controls <i>Hs</i>AQP8 or <i>At</i>TIP2;1, or <i>Hs</i>AQP1 or its mutants. The five amino acid residues of the extended selectivity filter in each construct are indicated in one letter code to the left in the order H2^P, LC^P, H5^P, LE^P, and HE^P, showing substitutions in bold. Complementation and failure to complement are indicated by + and −, respectively. Transformants were spotted at an OD₆₀₀ of 1 (right column) and 0.0001 (left column) on plates containing 0.2% proline or the indicated concentrations of ammonium as a sole nitrogen source and growth was recorded after 13 d at 28°C. Each panel showing growth at a specific concentration is compiled by individual pictures of each spot taken from a distinct growth condition. All single pictures were treated in the same manner.</p

Pore diameter and the extended selectivity filter.

<p>(A) Individual profiles of <i>At</i>TIP2;1 (green), glycerol-permeable <i>Ec</i>GlpF (blue), water-specific <i>So</i>PIP2;1 (closed conformation; purple), and <i>Hs</i>AQP4 (orange), as well as average diameter of five other open water-specific AQP structures (dashed line). Protein Data Bank IDs are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002411#pbio.1002411.s009" target="_blank">S1 Table</a>. NPA region and selectivity filter (SF) indicated by shading. In contrast to previously reported structures of AQPs, where the SF region constitutes the most narrow part of the channel, the pore diameter of <i>At</i>TIP2;1 is more uniform throughout the channel. (B) Graphic representation of a side view of the <i>At</i>TIP2;1 pore aligned with (A). The selectivity filter is highlighted by stick representation of residues in positions H2^P, HE^P, and LC^P (left to right). Nondisplayed residues at positions H5^P and LE^P are located in front of the visual plane. Close-up depicts electron density at 4σ. The high resolution of the structure makes it possible to pinpoint the nitrogen atoms in imidazole rings of histidines. The Nε of LC^P-His 131 forms a hydrogen bond (dashed yellow line) to a water molecule (Wat2) in the pore. (C) Vacuolar (top view of <i>At</i>TIP2;1) and corresponding extracellular view (<i>So</i>PIP2;1 and <i>Ec</i>GlpF) on the amino acid residues at the five positions (H2^P, LC^P, H5^P, LE^P, and HE^P) comprising the extended selectivity filter of the pore. <i>At</i>TIP2;1 (green) is compared to the water-specific <i>So</i>PIP2;1 (purple) and the glycerol-permeable <i>Ec</i>GlpF (blue). In <i>At</i>TIP2;1, histidines at H2^P and LC^P stabilize the arginine (Arg 200) at HE^P in a novel orientation, which is clearly different from its positioning in structures of water-specific and glycerol-permeable AQPs. The spatial orientation of the backbone carbonyls at position LE^P is similar in <i>At</i>TIP2;1 and <i>Ec</i>GlpF, whereas it deviates in the water-specific <i>So</i>PIP2;1. The Ile 185 at H5^P of <i>At</i>TIP2;1 results in a wider SF region compared to water-specific AQPs that have a histidine at this position. (D) The conservation of residues in the extended selectivity filter displayed in (C). The LC^P position that extends the selectivity filter is boxed in red. Plant TIPs and mammalian AQP8s are similar and distinctly different from water-specific AQPs in animals and plants (PIPs, plasma membrane intrinsic proteins), as well as the glycerol channel GlpF. Conservation patterns suggest a similar orientation of the conserved arginine at position HE^P of the selectivity filter of all TIPs and AQP8s, and furthermore that individual subgroups of TIPs and water-specific AQPs might have evolved specialized substrate profiles (details in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002411#pbio.1002411.s010" target="_blank">S2 Table</a>). Asterisk denotes identity to TIP2s, and colors highlight selectivity filters shown in (C).</p

Growth complementation of ammonium uptake-defective yeast strain by mutants of <i>At</i>TIP2;1.

<p>The 31019b yeast strain (<i>Δmep1–3</i>) was transformed with the empty vector pYeDP60u or with pYeDP60u-carrying cDNA encoding the positive controls <i>Hs</i>AQP8 or <i>Hs</i>AQP1, or <i>At</i>TIP2;1 or its mutants. The five amino acid residues of the extended selectivity filter in each construct are indicated in one letter code to the left in the order H2^P, LC^P, H5^P, LE^P, and HE^P, showing substitutions in bold. Complementation and failure to complement are indicated by + and −, respectively. Transformants were spotted at an OD₆₀₀ of 1 (right column) and 0.01 (left column) on plates containing 0.2% proline or the indicated concentrations of ammonium as a sole nitrogen source and growth was recorded after 13 d at 28°C. Each panel showing growth at a specific concentration is compiled by individual pictures of each spot taken from a distinct growth condition. All single pictures were treated in the same manner.</p

Ammonium accumulation and possible proton pathway.

<p>(A) MD simulations showing ammonium accumulation (blue mesh) at aspartate residues (yellow sticks) at vacuolar (top) and cytosolic (bottom) side of <i>At</i>TIP2;1. Water density (purple mesh) outlines the vertical main pore of the monomer and confirms existence of a water-filled side pore beneath loop C. Residues of the extended selectivity filter are depicted as sticks (H2^P-His 63 (blue), LC^P-His 131 (red), LE^P-Gly 194 (green), and HE^P-Arg 200 (brown)). (B) and (C) MD simulations demonstrating flexibility of His 131 at position LC^P being neutral (B) and positively charged (C). Color code as in (A). (D) Surface representation of the crystal structure depicting the water-filled side pore beneath loop C. Hydrogen bonds of water 10 (Wat10) as well as between Arg 200 at position HE^P in helix E and His 63 at position H2^P in helix 2 are indicated by dashed orange lines. (E) Tentative working model of ammonia-permeating <i>At</i>TIP2;1. Ammonium may contribute to ammonia permeation by accumulating on the vacuolar protein surface and by possibly having its protons shuttled back into the acidic vacuole by His 131 (red) at position LC^P in loop C via a water-filled side pore.</p

MD simulations of <i>At</i>TIP2;1.

<p>(A) The potential mean force (PMF) profiles for ammonia through <i>At</i>TIP2;1 (red) and through a model membrane containing 20% cholesterol (green). In the lower part of the panel, the number of hydrogen bonds between ammonia and <i>At</i>TIP2;1 are shown as function of position along the pore axis. Interactions with residues in the extended selectivity filter depicted in (C) are color-coded to resolve their contribution (H2^P-His 63 (blue), LC^P-His 131 (red), LE^P-Gly 194 (green) and HE^P-Arg 200 (brown)), and demonstrate hydrogen bonding to each of the four polar residues of the extended selectivity filter. (B) Snapshots of ammonia permeation. Cross section of <i>At</i>TIP2;1 shown as grey surface and green cartoon of the backbone. Side chains of selected amino acid residues in the selectivity filter are displayed as sticks and color coded as in (C). (C) Close-up of an ammonia molecule at the center, forming hydrogen bonds to four residues (H2^P-His 63, LC^P-His 131, LE^P-Gly 194, and HE^P-Arg 200) of the selectivity filter. The hydrogen bonds are indicated by orange dashes and distances are given in Å. Ile 185 at position H5^P of the selectivity filter, located in front of the visual plane, is not shown. The underlying data of panel A can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002411#pbio.1002411.s001" target="_blank">S1 Data</a>.</p

Topology and structure of <i>At</i>TIP2;1.

<p>(A) Topology plot showing membrane-spanning helices (H1–H6) and intervening loops (A–E). Homologous regions in the internal repeat are indicated by colors. The five positions of an extended selectivity filter are marked according to the key within the figure. The glycine (Gly 1) corresponding to the initiator methionine in <i>At</i>TIP2;1 is shaded in dark cyan next to a dashed line representing the TEV cleavage site, and the N-terminal deca-His tag is shaded in purple. (B) Two short helices (HB and HE) in loop B and E, connected via conserved NPA-motifs, form a seventh transmembrane segment. All membrane-spanning segments are tilted in the membrane, but it is most accentuated in helices H3, H6, HB, and HE facing the lipid bilayer. (C) Eight water molecules form a single file in the main pore of the monomer, connecting the cytosolic and vacuolar vestibules. At the top right, five additional water molecules are seen in a side pore underneath loop C. (D) <i>At</i>TIP2;1 tetramer viewed from the vacuolar side. Monomers are shown in surface representation and in the cartoon representation used in (B) and (C).</p

Jensen_Costa_Phospholipid_flipping_involves_a_central_cavity_in_P4_ATPases

The fileset includes:<div>- a structural model for the plant flippase ALA10 as a pdb file, with a PC headgroup docked into a central cavity</div><div>- Original flow cytometry data for all datasets used in Figures 2-6 of the manuscript. Each figure has been assigned a folder with the name of the figure the folder refers to. Inside each folder, subfolders with each individual experiment are included. A word file explains the content of each subfolder, including plasmids used for transformation and lipid tested. </div><div>- Original TLC plate scans used in Figure 2. These can be found in the folder corresponding to Figure 2. </div

The vast majority of Drs2p is in complex with Cdc50p, in a 1/1 stoichiometry.

<p><b>(A)</b> Yeasts were transformed either with a regular Bad-Drs2p/His₁₀-Cdc50p construct (with N-terminal tags) or with a related construct in which the TEV cleavage site between the His₁₀ tag and Cdc50p had been omitted. This resulted, after streptavidin-based purification, in either the classical sample (D–C) or in a sample where Cdc50p remains tagged with His₁₀ (D-_His10C). For both samples, the streptavidin-eluted fractions were diluted 5-fold (to about 70 µg/mL) in KNG buffer supplemented with 1 mg/mL DDM, 0.025 mg/mL PS and 0.025 mg/mL PI4P, and 300 µL of each diluted sample was mixed with 5 mg of dry Ni²⁺-TED resin (previously washed with the dilution buffer) and incubated on a wheel for 45 minutes in the cold room. Initial diluted samples (E_s), and unbound material (FT), were loaded onto a 10% SDS-PAGE and stained with silver nitrate. For D-His₁₀C, the E_s sample was further diluted 3-fold and 10-fold and aliquots (E_s/10 and E_s/3) were loaded for comparison with the FT sample. <b>(B)</b> 2.5 µg and 0.5 µg of streptavidin-purified Drs2p-Cdc50p complex were loaded onto a haloalkane-containing 4–20% gradient gel for both in-gel fluorescence analysis (left, Fluo) and subsequent Coomassie Blue staining of the same gel (right, CB).</p