Interactions between Transmembrane Helices within Monomers of the Aquaporin AtPIP2;1 Play a Crucial Role in Tetramer Formation

Abstract

Aquaporin (AQP) is a water channel protein found in various subcellular membranes of both prokaryotic and eukaryotic cells. The physiological functions of AQPs have been elucidated in many organisms. However, understanding their biogenesis remains elusive, particularly regarding how they assemble into tetramers. Here, we investigated the amino acid residues involved in the tetramer formation of the Arabidopsis plasma membrane AQP AtPIP2;1 using extensive amino acid substitution mutagenesis. The mutant proteins V41A/E44A, F51A/L52A, F87A/I91A, F92A/I93A, V95A/Y96A, and H216A/L217A, harboring alanine substitutions in the transmembrane (TM) helices of AtPIP2;1 polymerized into multiple oligomeric complexes with a variable number of subunits greater than four. Moreover, these mutant proteins failed to traffic to the plasma membrane, instead of accumulating in the endoplasmic reticulum (ER). Structure-based modeling revealed that these residues are largely involved in interactions between TM helices within monomers. These results suggest that inter-TM interactions occurring both within and between monomers play crucial roles in tetramer formation in the AtPIP2;1 complex. Moreover, the assembly of AtPIP2;1 tetramers is critical for their trafficking from the ER to the plasma membrane, as well as water permeability.

Cooperativity in Plant Plasma Membrane Intrinsic Proteins (PIPs): Mechanism of Increased Water Transport in Maize PIP1 Channels in Hetero-tetramers

“…It shows enhanced interactions with Ile-190, Thr-194, Phe-220 and Ala-221 from the TM4-TM5 region of ZmPIP1;2 (Figure 7A). Such extensive interactions are not possible for the small residue Ala. Based on the mutation studies, Yoo et al 50 have concluded that Ile-93 is important to stabilize the TM5 conformation at the monomer-monomer interface. However, they studied the importance of residues in tetramer formation of AtPIP2;1 homotetramers.…”

“…They concluded that the formation of homo- and hetero-tetramers can modulate the water permeability of the cell plasma membrane. Yoo et al 50 carried out extensive mutagenesis studies on Arabidopsis PIP2;1 to identify residues that could play important role in tetramer formation. Their studies indicated that residues in TM1, TM2 and TM5 involved in transmembrane helix-helix interactions within and between monomers are critical in tetramer assembly.…”

“…It has been shown that all hetero-tetrameric configurations (3:1, 2:2, 1:3) exist and all produced active channels localize in plasma membranes with equivalent efficiency in transporting water 49 . Similarly, a large number of mutants have been generated for the Arabidopsis PIP2;1 channel and certain residues within and between monomers have been found to be important for the heteromer formation and for the trafficking of the protein from endoplasmic reticulum to plasma membrane 50 . Modeling studies involving ZmPIP1;2 and ZmPIP2;5 hetero-tetramers have identified residues occurring at the monomer-monomer interface 51 .…”

Phaseolus vulgaris SUT1.1 is a high affinity sucrose‐proton co‐transporter

“…By using a protein known to be targeted to the plasma membrane (AtPIP2A; Yoo et al, 2016), it was resolved that PvSUT1.1 co-localizes with the AtPIP2A control, thus confirming that PvSUT1.1 is targeted to the plasma membrane. It is also apparent that GFP seems to also be present in the nuclei and the cytosol, which is where free GFP is usually found (Brandizzi et al, 2003).…”

“…A confocal microscope (Olympus FluoView TM FV1000) was used to take images of leaf epidermal cells two days after infiltration. As plasma membrane control, Arabidopsis PIP2A (At3g53420; Yoo et al, 2016) fused to mCherry (Shaner et al, 2004) was used.…”