Biomedical subjects

P Kjellbom

Publications and source records attributed to P Kjellbom.

12 recordsLinked to original sources

PubMed2000

Structural characterization of two aquaporins isolated from native spinach leaf plasma membranes.

Two members of the aquaporin family, PM28A and a new one, PM28C, were isolated and shown to be the major constituents of spinach leaf plasma membranes. These two isoforms were identified and characterized by matrix-assisted laser desorption ionization-mass spectrometry. Edman degradation yielded the amino acid sequence of two domains belonging to the new isoform. PM28B, a previously described isoform, was not found in our preparations. Scanning transmission electron microscopy mass analysis revealed both PM28 isoforms to be tetrameric. Two types of particles, a larger and a smaller one, were found by transmission electron microscopy of negatively stained solubilized proteins and by atomic force microscopy of PM28 two-dimensional crystals. The ratio of larger to smaller particles observed by transmission electron microscopy and single particle analysis correlated with the ratio of PM28A to PM28C determined by matrix-assisted laser desorption ionization-mass spectrometry. The absence of PM28B and the ratio of PM28A to PM28C indicate that these plasma membrane intrinsic proteins are differentially expressed in spinach leaves. These findings suggest that differential expression of the various aquaporin isoforms may regulate the water flux across the plasma membrane, in addition to the known mechanism of regulation by phosphorylation.

Amino Acid Sequence↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

An abundant TIP expressed in mature highly vacuolated cells.

Aquaporins are water channel proteins found in vacuolar membranes and plasma membranes, and belong to the major intrinsic protein (MIP) family of proteins. In the present study, we purified a 75 kDa MIP protein from a crude fraction of spinach leaf intracellular membranes. Upon urea/SDS-PAGE, the 75 kDa protein appeared as a 21 kDa polypeptide, and the 75 kDa species therefore probably represents a tetramer. The corresponding cDNA was obtained by PCR cloning and had an open reading frame encoding a 25.1 kDa protein. The protein, So-deltaTIP, was most homologous to the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Using affinity-purified So-deltaTIP-specific peptide antibodies, we investigated the subcellular and tissue distribution of So-deltaTIP. So-deltaTIP was specifically located in the vacuolar membrane. It was abundant in most vacuolated cells in all vegetative organs, but was excluded from the leaf epidermis as well as from the root phloem parenchyma and meristem. In spite of the high sequence homology between delta-TIPs of spinach, Arabidopsis, sunflower and radish, their expression patterns were totally different. However, a comparison of the expression pattern of So-deltaTIP with that of more distantly related TIPs showed similarities with Arabidopsis gamma-TIP, which is expressed in zones of cell elongation/differentiation but excluded from meristematic tissues. Meristematic cells are characterized by many small vacuoles as opposed to elongating and mature cells, which generally harbour a single, large vacuole. Our results indicate that the expression of So-deltaTIP may be induced when the large vacuole is formed.

Amino Acid Sequence↗

PubMed1999

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.

Journal Article↗

PubMed1998

Molecular characterization of a new type of receptor-like kinase (wlrk) gene family in wheat.

In plants, several types of receptor-like kinases (RLK) have been isolated and characterized based on the sequence of their extracellular domains. Some of these RLKs have been demonstrated to be involved in plant development or in the reaction to environmental signals. Here, we describe a RLK gene family in wheat (wlrk, wheat leaf rust kinase) with a new type of extracellular domain. A member of this new gene family has previously been shown to cosegregate with the leaf rust resistance gene Lr10. The diversity of the wlrk gene family was studied by cloning the extracellular domain of different members of the family. Sequence comparisons demonstrated that the extracellular domain consists of three very conserved regions interrupted by three variable regions. Linkage analysis indicated that the wlrk genes are specifically located on chromosome group 1 in wheat and on the corresponding chromosomes of other members of the Triticeae family. The wlrk genes are constitutively expressed in the aerial parts of the plant whereas no expression was detected in roots. Protein immunoblots demonstrated that the WLRK protein coded by the Lrk10 gene is an intrinsic plasma membrane protein. This is consistent with the hypothesis that WLRK proteins are receptor protein kinases localized to the cell surface. In addition, we present preliminary evidence that other disease resistance loci in wheat contain genes which are related to wlrk.

Amino Acid Sequence↗

PubMed1998

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.

Animals↗

PubMed1997

Oxidative cross-linking of plasma membrane arabinogalactan proteins.

Monoclonal antibodies which recognize carbohydrate in arabinogalactan proteins (AGPs) have revealed that certain carbohydrate epitopes at the outer plasma membrane surface are developmentally regulated. Some epitopes are expressed according to cell position, and AGPs are thought to play a role in cell-cell interaction during development. This study demonstrates that sugar beet plasma membranes contain two subfamilies of AGPs, with apparent molecular masses of 82 and 97 kDa, and that each subfamily consists of a small number of acidic AGP isoforms. Excision of leaves generates three additional AGP complexes with apparent molecular masses of 120, 170 and 210 kDa, with the 170 kDa complex being the major form induced by excision. The addition of millimolar concentrations of H2O2 to a partially purified fraction of the 82 and 97 kDa AGPs also generates AGP complexes, with the 170 kDa complex as the major form. These results indicate that the plasma membrane AGPs are a target for endogenous H2O2.

Cell Membrane↗

PubMed1996

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+.

Amino Acid Sequence↗

PubMed1995

Cell-context signalling.

In plants, cells differentiate according to their position with relation to their cell neighbours. Monoclonal antibody (MAb) probes to polysaccharide epitopes, present at the surfaces of all plant cells, have defined a family of proteoglycan antigens which signify cellular position. These MAbs have been used to sort the single cells present in carrot somatic cell cultures on the basis of the presence or absence of specific polysaccharide epitopes. This sorting allows embryo initial cells to be cultured among different cell collectives (based on their polysaccharide epitope expression) and thus in altered contextual backgrounds. These experiments have shown that specific populations of embryo initial precursor cells induce and sustain the early development of the embryo initials, revealing that the populations of different cell collectives which are defined by different polysaccharide epitopes (cell-context) serves important regulatory function in early plant development. Somatic embryo initials deprived of the influence of the cell collective-defined by the presence of the polysaccharide epitope recognised by the MAb JIM8-establish unorganised first divisions and develop as callus. However, in the presence of the JIM8-reactive cell collective, or medium conditioned by the collective, the initials develop into somatic embryos. This demonstrates that the cells defined by the JIM8 polysaccharide epitope are necessary to sustain the meristematic activity which drives the renewed development. Transfer of a cell-wall signal from the JIM8-reactive cells to cellular situations in carrot seedlings in which they would not normally occur (out-of-context signals) stimulates lateral root production, thus demonstrating that the inductive signal operative in suspension cultures can be reinterpreted by specific cells later in development and reinitiate meristematic activity. The communication between the precursor cells defined by JIM8 and embryo initials defines an early cell-cell interaction in developing carrot plants. Labelling of flower sections suggests that the same interaction exists between embryo apical and basal cells early in normal development.

Cell Communication↗

PubMed1995

Identification of cDNA clones encoding valosin-containing protein and other plant plasma membrane-associated proteins by a general immunoscreening strategy.

An approach was developed for the isolation and characterization of soybean plasma membrane-associated proteins by immunoscreening of a cDNA expression library. An antiserum was raised against purified plasma membrane vesicles. In a differential screening of approximately 500,000 plaque-forming units with the anti-(plasma membrane) serum and DNA probes derived from highly abundant clones isolated in a preliminary screening, 261 clones were selected from approximately 1,200 antiserum-positive plaques. These clones were classified into 40 groups by hybridization analysis and 5'- and 3'-terminal sequencing. By searching nucleic acid and protein sequence data bases, 11 groups of cDNAs were identified, among which valosin-containing protein (VCP), clathrin heavy chain, phospholipase C, and S-adenosylmethionine:delta 24-sterol-C-methyltransferase have not to date been cloned from plants. The remaining 29 groups did not match any current data base entries and may, therefore, represent additional or yet uncharacterized genes. A full-length cDNA encoding the soybean VCP was sequenced. The high level of amino acid identity with vertebrate VCP and yeast CDC48 protein indicates that the soybean protein is a plant homolog of vertebrate VCP and yeast CDC48 protein.

Amino Acid Sequence↗

PubMed1992

Elicitor- and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response.

Treatment of bean or soybean cells with fungal elicitor or glutathione causes a rapid insolubilization of preexisting (hydroxy)proline-rich structural proteins in the cell wall. This insolubilization, which involves H2O2-mediated oxidative cross-linking, is initiated within 2 min and is complete within 10 min under optimal conditions, and hence, precedes the expression of transcription-dependent defenses. Cross-linking is also under developmental control during hypocotyl growth and in tissues subject to mechanical stress such as the stem-petiole junction. Stimulus-dependent oxidative cross-linking of wall structural proteins is a novel site of cellular regulation with potentially important functions in cell maturation and toughening of cell walls in the initial stages of plant defense.

Amino Acid Sequence↗

PubMed1987

Lipid composition of plasma membranes isolated from light-grown barley (Hordeum vulgare) leaves: identification of cerebroside as a major component.

The total lipid composition of highly purified plasma membranes from light-grown barley (Hordeum vulgare) leaves was investigated. The plasma membranes were separated from intracellular membranes by subfractionation of the microsomal fraction using aqueous polymer two-phase partitioning. A novel finding was that glucocerebroside was a major lipid of the plasma membrane (23 mol%). The most abundant lipid class in the plasma membrane was phospholipid (42 mol%), consisting mainly of phosphatidylcholine and phosphatidylethanolamine, together with free sterols at a level of 28 mol%. The only free sterols of the plasma membrane were campesterol (15%), stigmasterol (23%), and sitosterol (62%). The plasma membrane contained a relatively high proportion of saturated fatty acids compared to the bulk of intracellular membranes, the major components of the plasma membrane being palmitic (16:0), linoleic (18:2), and linolenic (18:3) acids in approximately equal amounts.

Cell Membrane↗