Per Kjellbom

Structural mechanism of plant aquaporin gating

Plants counteract fluctuations in water supply by regulating all aquaporins in the cell plasma membrane. Channel closure results either from the dephosphorylation of two conserved serine residues under conditions of drought stress, or from the protonation of a conserved histidine residue following a drop in cytoplasmic pH due to anoxia during flooding. Here we report the X-ray structure of the spinach plasma membrane aquaporin SoPIP2;1 in its closed conformation at 2.1 Å resolution and in its open conformation at 3.9 Å resolution, and molecular dynamics simulations of the initial events governing gating. In the closed conformation loop D caps the channel from the cytoplasm and thereby occludes the pore. In the open conformation loop D is displaced up to 16 Å and this movement opens a hydrophobic gate blocking the channel entrance from the cytoplasm. These results reveal a molecular gating mechanism which appears conserved throughout all plant plasma membrane aquaporins.

Water Transport Activity of the Plasma Membrane Aquaporin PM28A Is Regulated by Phosphorylation

PM28A is a major intrinsic protein of the spinach leaf plasma membrane and the major phosphoprotein. Phosphorylation of PM28A is dependent in vivo on the apoplastic water potential and in vitro on submicromolar concentrations of Ca2+. Here, we demonstrate that PM28A is an aquaporin and that its water channel activity is regulated by phosphorylation. Wild-type and mutant forms of PM28A, in which putative phosphorylation sites had been knocked out, were expressed in Xenopus oocytes, and the resulting increase in osmotic water permeability was measured in the presence or absence of an inhibitor of protein kinases (K252a) or of an inhibitor of protein phosphatases (okadaic acid). The results indicate that the water channel activity of PM28A is regulated by phosphorylation of two serine residues, Ser-115 in the first cytoplasmic loop and Ser-274 in the C-terminal region. Labeling of spinach leaves with 32P-orthophosphate and subsequent sequencing of PM28A-derived peptides demonstrated that Ser-274 is phosphorylated in vivo, whereas phosphorylation of Ser-115, a residue conserved among all plant plasma membrane aquaporins, could not be demonstrated. This identifies Ser-274 of PM28A as the amino acid residue being phosphorylated in vivo in response to increasing apoplastic water potential and dephosphorylated in response to decreasing water potential. Taken together, our results suggest an active role for PM28A in maintaining cellular water balance.

[52] Preparation of high-purity plasma membranes

Publisher Summary This chapter describes a method using the preparation of plasma membranes from light-grown oat ( Arena sativa L. ) leaves as an example. Using the batch procedure, two fractions containing purified plasma membrane (U 3 and U 3 , ) and one fraction containing intracellular membranes depleted of plasma membrane (L 1 ) are obtained. Specific staining with phosphotungstic acid or silicotungstic acid seems to be the only universal marker for the plant plasma membrane and the only one that permits a real estimation of the purity of the preparations. Based on this staining the purity is estimated to be higher than 90% and often close to 100%, plasma membrane and similar purities are reported for plasma membrane from maize roots. (>90%) for plasma membrane preparations obtained both by phase partitioning and free flow electrophoresis.

The role of aquaporins in cellular and whole plant water balance

Aquaporins are water channel proteins belonging to the major intrinsic protein (MIP) superfamily of membrane proteins. More than 150 MIPs have been identified in organisms ranging from bacteria to animals and plants. In plants, aquaporins are present in the plasma membrane and in the vacuolar membrane where they are abundant constituents. Functional studies of aquaporins have hitherto mainly been performed by heterologous expression in Xenopus oocytes. A main issue is now to understand their role in the plant, where they are likely to be important both at the cellular and at the whole plant level. Plants contain a large number of aquaporin isoforms with distinct cell type- and tissue-specific expression patterns. Some of these are constitutively expressed, whereas the expression of others is regulated in response to environmental factors, such as drought and salinity. At the protein level, regulation of water transport activity by phosphorylation has been reported for some aquaporins.

Whole gene family expression and drought stress regulation of aquaporins

Since many aquaporins (AQPs) act as water channels, they are thought to play an important role in plant water relations. It is therefore of interest to study the expression patterns of AQP isoforms in order to further elucidate their involvement in plant water transport. We have monitored the expression patterns of all 35 Arabidopsis AQPs in leaves, roots and flowers by cDNA microarrays, specially designed for AQPs, and by quantitative real-time reverse transcriptase PCR (Q-RT-PCR). This showed that many AQPs are pre-dominantly expressed in either root or flower organs, whereas no AQP isoform seem to be leaf specific. Looking at the AQP subfamilies, most plasma membrane intrinsic proteins (PIPs) and some tonoplast intrinsic proteins (TIPs) have a high level of expression, while NOD26-like proteins (NIPs) are present at a much lower level. In addition, we show that PIP transcripts are generally down-regulated upon gradual drought stress in leaves, with the exception of AtPIP1;4 and AtPIP2;5, which are up-regulated. AtPIP2;6 and AtSIP1;1 are constitutively expressed and not significantly affected by the drought stress. The transcriptional down-regulation of PIP genes upon drought stress could also be observed on the protein level.

The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca2+ and apoplastic water potential.

We show that homologs of the major intrinsic protein (MIP) family are major integral proteins of the spinach leaf plasma membrane and constitute approximately 20% of integral plasma membrane protein. By using oligonucleotide primers based on partial amino acid sequences for polymerase chain reaction and screening of a spinach leaf cDNA library, we obtained two full-length clones of MIP homologs (pm28a and pm28b). One of these clones, pm28a, was sequenced, and it encodes a protein (PM28A) of 281 amino acids with a molecular mass of 29.9 kD. DNA gel blots indicated that PM28A is the product of a single gene, and RNA gel blots showed that pm28a is ubiquitously expressed in the plant. In vivo phosphorylation of the 28-kD polypeptide(s), corresponding to PM28A and PM28B, was dependent on apoplastic water potential, suggesting a role in regulation of cell turgor for these putative aquaporins. In vitro, only one of the homologs, PM28A, was phosphorylated. Phosphorylation of PM28A occurred on Ser-274, seven amino acids from the C terminus of the protein, within a consensus phosphorylation site (Ser-X-Arg) for vertebrate protein kinase C. In vitro phosphorylation of PM28A was due to a plasma membrane-associated protein kinase and was strictly dependent on submicromolar concentrations of Ca2+.

Aquaporins and water homeostasis in plants

Aquaporins are water channel proteins of vacuolar and plasma membranes. When opened they facilitate the passive movement of water molecules down a water potential gradient. In Arabidopsis, 30 genes have been found that code for aquaporin homologues. Some of these genes code for highly abundant constitutively expressed proteins and some are known to be temporally and spatially regulated during development and in response to stress. The water transport activity of two aquaporins is regulated at the protein level by phosphorylation and dephosphorylation. At a given time, cells express several different aquaporins, and it is probable that vacuolar and plasma membrane aquaporins acting in concert are responsible for the cytosolic osmoregulation that is necessary for maintaining normal metabolic processes. Inhibition studies of aquaporins in vivo and antisense mutant studies suggest that, in addition to cytosolic osmoregulation, aquaporins are important for the bulk flow of water in plants.

Sidedness of plant plasma membrane vesicles purified by partitioning in aqueous two-phase systems

Preparations of plant plasma membrane vesicles were obtained by partition in dextran‐polyethylene glycol two‐phase systems. By this procedure particles are separated according to their surface properties, and an iso‐osmotic environment is maintained throughout. The vesicles thus produced are right‐side‐out and sealed, as measured by enzyme [(K+ + Mg2+)‐ATPase and glucan synthetase II] latency on addition of Triton X‐100.

High-resolution x-ray structure of human aquaporin 5

Human aquaporin 5 (HsAQP5) facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-Å resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.

Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris

The aquaporin PM28A is one of the major integral proteins in spinach leaf plasma membranes. Phosphorylation/dephosphorylation of Ser274 at the C‐terminus and of Ser115 in the first cytoplasmic loop has been shown to regulate the water channel activity of PM28A when expressed in Xenopus oocytes. To understand the mechanisms of the phosphorylation‐mediated gating of the channel the structure of PM28A is required. In a first step we have used the methylotrophic yeast Pichia pastoris for expression of the pm28a gene. The expressed protein has a molecular mass of 32462 Da as determined by matrix‐assisted laser desorption ionization‐mass spectrometry, forms tetramers as revealed by electron microscopy and is functionally active when reconstituted in proteoliposomes. PM28A was efficiently solubilized from urea‐ and alkali‐stripped Pichia membranes by octyl‐β‐D‐thioglucopyranoside resulting in a final yield of 25 mg of purified protein per liter of cell culture.