Jozefkowicz C et al. (2013), Loop A is critical for the functional interacti...

XB-ART-48180

PLoS One 2013 Jan 01;83:e57993. doi: 10.1371/journal.pone.0057993.

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Loop A is critical for the functional interaction of two Beta vulgaris PIP aquaporins.

Jozefkowicz C , Rosi P , Sigaut L , Soto G , Pietrasanta LI , Amodeo G , Alleva K .

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Research done in the last years strongly support the hypothesis that PIP aquaporin can form heterooligomeric assemblies, specially combining PIP2 monomers with PIP1 monomers. Nevertheless, the structural elements involved in the ruling of homo versus heterooligomeric organization are not completely elucidated. In this work we unveil some features of monomer-monomer interaction in Beta vulgaris PIP aquaporins. Our results show that while BvPIP2;2 is able to interact with BvPIP1;1, BvPIP2;1 shows no functional interaction. The lack of functional interaction between BvPIP2;1 and BvPIP1;1 was further corroborated by dose-response curves of water permeability due to aquaporin activity exposed to different acidic conditions. We also found that BvPIP2;1 is unable to translocate BvPIP1;1-ECFP from an intracellular position to the plasma membrane when co-expressed, as BvPIP2;2 does. Moreover we postulate that the first extracellular loop (loop A) of BvPIP2;1, could be relevant for the functional interaction with BvPIP1;1. Thus, we investigate BvPIP2;1 loop A at an atomic level by Molecular Dynamics Simulation (MDS) and by direct mutagenesis. We found that, within the tetramer, each loop A presents a dissimilar behavior. Besides, BvPIP2;1 loop A mutants restore functional interaction with BvPIP1;1. This work is a contribution to unravel how PIP2 and PIP1 interact to form functional heterooligomeric assemblies. We postulate that BvPIP2;1 loop A is relevant for the lack of functional interaction with BvPIP1;1 and that the monomer composition of PIP assemblies determines their functional properties.

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Species referenced: Xenopus laevis
Genes referenced: gnl3 pafah1b1

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	Figure 2. Localization of BvPIP1;1-ECFP and BvPIP2;2-EYFP in Xenopus laevis oocytes.Radial (x–z) confocal images of X. laevis oocytes expressing BvPIP1;1-ECFP (A) (green) and BvPIP2;2- EYFP (C) (green), previously injected with TMR-Dextran (red). The oocyte surface is on the right of each image frame and the interior of the oocyte is to the left. Inside each image the enlargement of the indicated square section is shown. Confocal (x–y) images collected at various focal depths into the X. laevis oocyte expressing BvPIP1;1-ECFP (B) and BvPIP2;2-EYFP (D) at 1µm steps from outside the oocyte till the cortical granules level, approximately 5 µm from the plasma membrane, are shown.
	Figure 3. Localization of BvPIP1;1-ECFP when co-expressed with BvPIP2;2 or BvPIP2;1 in Xenopus laevis oocytes.Radial (x–z) confocal images of X. laevis oocytes co-expressing BvPIP1;1-ECFP:BvPIP2;2 (green) (A) or co-expressing BvPIP1;1-ECFP:BvPIP2;1 (green) (D), both previously injected with TMR-Dextran (red). The oocyte surface is near the top of each image frame and the interior of the oocyte is in the bottom. Inside the image the enlargement of the indicated square section is shown. Stack of confocal (x–y) images were collected at various focal depths into the oocyte and then deconvolved and surface-render reconstructed with Huygens Professional Software. (B) Projections of the z-stack of images acquired with 100 nm step for oocytes co-expressing BvPIP1;1-ECFP:BvPIP2;2 (green) and (E) for oocytes co-expressing BvPIP1;1-ECFP:BvPIP2;1(green). (C) and (F) shows several views of the 3D reconstructed images for oocytes co-expressing BvPIP1;1-ECFP:BvPIP2;2 (green) and BvPIP1;1-ECFP:BvPIP2;1(green), respectively. The 0° view corresponds to the cortical granules level inside the oocyte and 180° to the plasma membrane plane (approximately 5 µm from the cortical granules level).
	Figure 4. Effect of pH on oocytes membrane Pf expressing BvPIP2;1 alone or co-expressing BvPIP2;1 with BvPIP1;1.To evaluate cytosolic pH sensing, oocytes expressing BvPIP2;1, co-expressing BvPIP2;1-BvPIP1;1 in a 1∶1 mass ratio or non-injected (NI) were incubated 15 min at different pH media. Then each oocyte was transferred to a fivefold-diluted medium at the same pH and the swelling assay was performed as described in Materials and Methods. Three independent experiments were performed and for each pH condition 7–10 oocytes were tested. The main figure shows representative values obtained on a same batch of oocytes (mean Pf ±SEM). Data were fitted to a sigmoidal dose-response curve using Graph Pad Prism (version 3.02). The inset shows mean EC50±SEM, n = 3 independent experiments; EC50 are not significantly different (p>0.05).
	Figure 5. Conserved residues of reported PIP2 that interact with PIP1.Alignment of the PIP consensus sequences between the last aminoacid of the first TMH-transmembrane helix- and the first residue of the second TMH, including loop A (residues 64–72). The PIP consensus sequence is based on PIP2 that interact with PIP1. Sequence conservation is displayed by the sequence logos technique. The corresponding residues in BvPIP2;1 primary sequence, according to this alignment, are shown down the logo. In red are indicated the selected residues to be mutated.
	Figure 6. BvPIP2;1 loops A MDS frames superimposition.The figure shows the MDS for BvPIP2;1 loops A. Each BvPIP2;1 monomer is in a different color; chain A is in yellow, chain B is in green, chain C is in blue and chain D is in red. The superimposition of 30 ns MDS of BvPIP2;1 loops A is shown in a color range, where red is the starting position, white is an intermediate position and blue is the final one.
	Figure 7. BvPIP2;1 loop A root mean square fluctuation (RMSF).The RMSF of loops A are shown in yellow, green, blue and red lines corresponding to chains A, B, C and D respectively in all panels. Panels from A to C represent temporal intervals of the MDS ranging from 0 to 10 ns, 10 to 20 ns and 20 to 30 ns respectively. The inset shows the BvPIP2;1 model, where green is used to point extracellular elements of the aquaporin, blue to point intracellular elements, purple to mark alpha helixes, bordeaux to distinguish loops B and E embedded in the membrane region and finally grey to show N-t and C-t. The same color pattern is used in the x-axis of panels to discriminate the location of residues of the primary structure in the protein. In the inset a red arrow is used to point loop A. The figure shows valleys in the RMSF which points that the secondary structure remains stable, the peaks represent movable parts of the protein and comparing extracellular elements, loop A is most flexible than loop C along the whole MDS.
	Figure 8. Osmotic permeability (Pf) of oocytes membranes expressing BvPIP2;1 mutants (N64H/E65Q or N64I/E65Q) with BvPIP1;1.BvPIP2;1 mutants (N64H/E65Q and N64I/E65Q) show high water transport activity indicating that they are functional aquaporins. Both mutants are able to functionally interact with BvPIP1;1 as they generate oocyte membrane Pf values far superior to those promoted by the mutant alone (*p<0.001). NI are non-injected oocytes. Values are representative data of three independent experiments using different oocyte batches. For each condition mean values are shown as mean Pf ±SEM, n = 12−15.
	Figure 9. pH dose-response curve of BvPIP2;1 N64I/E65Q mutant and its co-injection with BvPIP1;1.The effect of cytosolic acidification on Pf was tested on oocytes injected with BvPIP2;1 N64I/E65Q cRNA alone or co-injected with BvPIP1;1 cRNA in a 1∶1 mass ratio. Co-expression of BvPIP2;1 N64I/E65Q:BvPIP1;1 account for a different pH sensitivity in comparison with BvPIP2;1 N64I/E65Q alone. In the main figure values are representative data from three independent experiments using different oocyte batches. For each condition mean values are shown as Pf ± SEM, n = 7−10. Data were fitted to a sigmoidal dose-response curve using Graph Pad Prism (version 3.02). The inset shows mean EC50±SEM, n = 3 independent experiments, EC50 are significantly different (*p<0.05).
	Figure 1. Effect of BvPIP2;1 and BvPIP1;1 co-expression on oocyte plasma membrane permeability (Pf).Different amounts of cRNA of BvPIP2;1, BvPIP1;1 or a mix of BvPIP2;1 and BvPIP1;1 (BvPIP2;1:BvPIP1;1) were injected in Xenopus oocytes and after three days osmotic water permeability coefficient (Pf) was determined. In brackets is the relative quantity of cRNA injected in each oocyte, being 1 equal to 0,3 ng, and 2 or 3, two or three fold that amount, respectively. A four-fold injection of BvPIP2;1 (4) was used as a control to show that the expression system was not saturated, NI are non-injected oocytes. Data are expressed as mean values (mean Pf ±SEM, n = 12−15). The figure shows representative data from five independent experiments. Different letters indicate significance between bars (p<0.05). All Pf corresponding to oocytes co-injected with different cRNA ratios of BvPIP2;1: BvPIP1;1 were not significantly different from Pf of BvPIP2;1 (1) injected oocytes.

References [+] :

Agre, Functional analyses of aquaporin water channel proteins. 1999, Pubmed, Xenbase