Daniel M. Roberts

Homology modeling of representative subfamilies of Arabidopsis major intrinsic proteins. Classification based on the aromatic/arginine selectivity filter.

Major intrinsic proteins (MIPs) are a family of membrane channels that facilitate the bidirectional transport of water and small uncharged solutes such as glycerol. The 35 full-length members of the MIP family in Arabidopsis are segregated into four structurally homologous subfamilies: plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), nodulin 26-like intrinsic membrane proteins (NIPs), and small basic intrinsic proteins (SIPs). Computational methods were used to construct structural models of the putative pore regions of various plant MIPs based on homology modeling with the atomic resolution crystal structures of mammalian aquaporin 1 and the bacterial glycerol permease GlpF. Based on comparisons of the narrow selectivity filter regions (the aromatic/Arg [ar/R] filter), the members of the four phylogenetic subfamilies of Arabidopsis MIPs can be classified into eight groups. PIPs possess a uniform ar/R signature characteristic of high water transport aquaporins, whereas TIPs are highly diverse with three separate conserved ar/R regions. NIPs possess two separate conserved ar/R regions, one that is similar to the archetype, soybean (Glycine max) nodulin 26, and another that is characteristic of Arabidopsis NIP6;1. The SIP subfamily possesses two ar/R subgroups, characteristic of either SIP1 or SIP2. Both SIP ar/R residues are divergent from all other MIPs in plants and other kingdoms. Overall, these findings suggest that higher plant MIPs have a common fold but show distinct differences in proposed pore apertures, potential to form hydrogen bonds with transported molecules, and amphiphilicity that likely results in divergent transport selectivities.

Structure, function, and mechanism of action of Calmodulin

The structure, function, and mechanism of action of plant calmodulins have been the subject of intense study for plant scientists during the past several years. While precedents in animal biochemistry and physiology have suggested logical starting points for studies of how calmodulin is involved in higher plant and algal cell function, recent biochemical studies have demonstrated unique structural characteristics for this highly conserved protein. Enzym‐ological analyses have demonstrated novel functional properties and provided limited insight into the molecular mechanisms of calmodulin action. This review will summarize much of the early work, but will concern itself mainly with the methods and approaches that are used to study plant and animal calmodulins as well as models for how calmodulin may be involved in plant cell function.

Voltage-Dependent Cation Channels Permeable to NH4+, K+, and Ca2+ in the Symbiosome Membrane of the Model Legume Lotus japonicus

The symbiosome of nitrogen fixing root nodules mediates metabolite exchange between endosymbiotic rhizobia bacteria and the legume host. In the present study, the ion currents of the symbiosome membrane of the model legume Lotus japonicus were analyzed by patch-clamp recording. Both excised and symbiosome-attached patches exhibited a large inward (toward the cytosolic side of the membrane) current that is activated in a time-dependent manner by negative (on the cytosolic side) potentials. Based on reversal potential determinations and recordings with the impermeant cationN-methyl-glucamine, this current shows a high permeability for monovalent cations with no apparent permeability for anions. The current also showed a finite Ca2+ permeability. However, the currents were predominantly carried by univalent cations with a slightly greater selectivity for NH4 +over K+. Increased Ca2+ concentration inhibited the current with a K 0.5 for inhibition of 0.317 mm. The current showed strong rectification that is mediated by divalent cations (either Mg2+ or Ca2+). The influence of divalent cations is symmetrical in nature, because rectification can be exerted in either direction depending upon which side of the membrane has the highest concentration of divalent cations. However, based on observations with symbiosome-attached patches, the direction of the current in vivo is proposed to be toward the cytosol with cytosolic Mg2+acting as the putative gating regulator. The findings suggest thatL. japonicus possesses a voltage-dependent cation efflux channel that is capable of exporting fixed NH4 +, and may also play an additional role in Ca2+ transport.

Interaction of cytosolic glutamine synthetase of soybean root nodules with the C-terminal domain of the symbiosome membrane nodulin 26 aquaglyceroporin

Nodulin 26 (nod26) is a major intrinsic protein that constitutes the major protein component on the symbiosome membrane (SM) of N2-fixing soybean nodules. Functionally, nod26 forms a low energy transport pathway for water, osmolytes, and NH3 across the SM. Besides their transport functions, emerging evidence suggests that high concentrations of major intrinsic proteins on membranes provide interaction and docking targets for various cytosolic proteins. Here it is shown that the C-terminal domain peptide of nod26 interacts with a 40-kDa protein from soybean nodule extracts, which was identified as soybean cytosolic glutamine synthetase GS1β1 by mass spectrometry. Fluorescence spectroscopy assays show that recombinant soybean GS1β1 binds the nod26 C-terminal domain with a 1:1 stoichiometry (Kd = 266 nm). GS1β1 also binds to isolated SMs, and this binding can be blocked by preincubation with the C-terminal peptide of nod26. In vivo experiments using either a split ubiquitin yeast two-hybrid system or bimolecular fluorescence complementation show that the four cytosolic GS isoforms expressed in soybean nodules interact with full-length nod26. The binding of GS, the principal ammonia assimilatory enzyme, to the conserved C-terminal domain of nod26, a transporter of NH3, is proposed to promote efficient assimilation of fixed nitrogen, as well as prevent potential ammonia toxicity, by localizing the enzyme to the cytosolic side of the symbiosome membrane.

GmN70 and LjN70. Anion Transporters of the Symbiosome Membrane of Nodules with a Transport Preference for Nitrate

A cDNA was isolated from soybean (Glycine max) nodules that encodes a putative transporter (GmN70) of the major facilitator superfamily. GmN70 is expressed predominantly in mature nitrogen-fixing root nodules. By western-blot and immunocytochemical analyses, GmN70 was localized to the symbiosome membrane of infected root nodule cells, suggesting a transport role in symbiosis. To investigate its transport function, cRNA encoding GmN70 was expressed in Xenopus laevis oocytes, and two-electrode voltage clamp analysis was performed. Ooctyes expressing GmN70 showed outward currents that are carried by anions with a selectivity of nitrate > nitrite ≫ chloride. These currents showed little sensitivity to pH or the nature of the counter cation in the oocyte bath solution. One-half maximal currents were induced by nitrate concentrations between 1 to 3 mm. No apparent transport of organic anions was observed. Voltage clamp records of an ortholog of GmN70 from Lotus japonicus (LjN70; K. Szczyglowski, P. Kapranov, D. Hamburger, F.J. de Bruijn [1998] Plant Mol Biol 37: 651–661) also showed anion currents with a similar selectivity profile. Overall, these findings suggest that GmN70 and LjN70 are inorganic anion transporters of the symbiosome membrane with enhanced preference for nitrate. These transport activities may aid in regulation of ion and membrane potential homeostasis, possibly in response to external nitrate concentrations that are known to regulate the symbiosis.

[24] The use of synthetic oligodeoxyribonucleotides in the examination of calmodulin gene and protein structure and function

Publisher Summary This chapter discusses how synthetic oligonucleotides are used in studies of calmodulin and related calcium-binding proteins. The focus is on two specific applications of synthetic oligonucleotides: site-specific mutagenesis and detection of recombinant DNA vectors containing DNA inserts coding for calmodulin-like structures. The database of information on the phylogeny and ontogeny of calmodulin provides the necessary background for the initiation of studies utilizing a direct approach to structure and function that combines recombinant DNA, comparative enzymology, and protein chemistry techniques. The synthetic calmodulin gene was designed for mutagenesis by two approaches: cassette-based and M13-based mutagenesis. Mutagenesis is performed on a construction consisting of the synthetic calmodulin gene cloned in the EcoRI and BamHI sites of pUC8. Once the mutant calmodulin gene sequence is generated and verified, an expression vector containing the mutated gene sequences is constructed. Calmodulin mutant genes are expressed by cloning on the expression plasmid pKK223-3. The final purified product is characterized by a variety of analytical techniques including SDS-polyacrylamide gel electrophoresis, amino acid composition analyses of acid hydrolysates of the whole protein and selected peptide fractions, and limited amino acid sequence analyses.

Molecular Mechanisms of Calmodulin Action

Calcium has many biological functions and several groups of macromolecules have the ability to bind calcium with various selectivities and affinities. A rapidly expanding body of knowledge about the structure, thermodynamic and kinetic properties, and cell biology of calcium binding proteins strongly indicates that the targets or receptors for calcium acting as a signal transducer in eukaryotic cells are a class of calcium binding proteins referred to as calcium modulated proteins. These proteins are characterized by their ability to bind calcium in a reversible manner with dissociation constants in the nanomolar to micromolar range under physiological conditions. Although it is not possible yet to predict with any degree of certainty what type of calcium binding structure or molecular mechanism might be associated with a given biological function of calcium, a trend has begun to emerge from the detailed analyses of calcium modulated proteins1