Tetramerization dynamics of C-terminal domain underlies isoform-specific cAMP gating in hyperpolarization-activated cyclic nucleotide-gated channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are dually activated by hyperpolarization and binding of cAMP to their cyclic nucleotide binding domain (CNBD). HCN isoforms respond differently to cAMP; binding of cAMP shifts activation of HCN2 and HCN4 by 17 mV but shifts that of HCN1 by only 2-4 mV. To explain the peculiarity of HCN1, we solved the crystal structures and performed a biochemical-biophysical characterization of the C-terminal domain (C-linker plus CNBD) of the three isoforms. Our main finding is that tetramerization of the C-terminal domain of HCN1 occurs at basal cAMP concentrations, whereas those of HCN2 and HCN4 require cAMP saturating levels. Therefore, HCN1 responds less markedly than HCN2 and HCN4 to cAMP increase because its CNBD is already partly tetrameric. This is confirmed by voltage clamp experiments showing that the right-shifted position of V(½) in HCN1 is correlated with its propensity to tetramerize in vitro. These data underscore that ligand-induced CNBD tetramerization removes tonic inhibition from the pore of HCN channels.

Characterization of a carbohydrate transporter from symbiotic glomeromycotan fungi.

The symbiotic relationships between mycorrhizal fungi and plants have an enormous impact on terrestrial ecosystems. Most common are the arbuscular mycorrhizas, formed by fungi belonging to the phylum Glomeromycota. Arbuscular mycorrhizal fungi facilitate the uptake of soil nutrients by plants and in exchange obtain carbohydrates, thus representing a large sink for atmospheric plant-fixed CO(2). However, how carbohydrates are transported through the symbiotic interface is still unknown. Here we report the characterization of the first known glomeromycotan monosaccharide transporter, GpMST1, by exploiting the unique symbiosis of a glomeromycotan fungus (Geosiphon pyriformis) with cyanobacteria. The GpMST1 gene has a very low GC content and contains six introns with unusual boundaries. GpMST1 possesses twelve predicted transmembrane domains and functions as a proton co-transporter with highest affinity for glucose, then mannose, galactose and fructose. It belongs to an as yet uncharacterized phylogenetic monosaccharide transporter clade. This initial characterization of a new transporter family involved in fungal symbiosis will lead to a better understanding of carbon flows in terrestrial environments.

A functional calcium-transporting ATPase encoded by chlorella viruses.

Calcium-transporting ATPases (Ca(2+) pumps) are major players in maintaining calcium homeostasis in the cell and have been detected in all cellular organisms. Here, we report the identification of two putative Ca(2+) pumps, M535L and C785L, encoded by chlorella viruses MT325 and AR158, respectively, and the functional characterization of M535L. Phylogenetic and sequence analyses place the viral proteins in group IIB of P-type ATPases even though they lack a typical feature of this class, a calmodulin-binding domain. A Ca(2+) pump gene is present in 45 of 47 viruses tested and is transcribed during virus infection. Complementation analysis of the triple yeast mutant K616 confirmed that M535L transports calcium ions and, unusually for group IIB pumps, also manganese ions. In vitro assays show basal ATPase activity. This activity is inhibited by vanadate, but, unlike that of other Ca(2+) pumps, is not significantly stimulated by either calcium or manganese. The enzyme forms a (32)P-phosphorylated intermediate, which is inhibited by vanadate and not stimulated by the transported substrate Ca(2+), thus confirming the peculiar properties of this viral pump. To our knowledge this is the first report of a functional P-type Ca(2+)-transporting ATPase encoded by a virus.

Arbuscular mycorrhiza: studies on the geosiphon symbiosis lead to the characterization of the first glomeromycotan sugar transporter.

The intimate arbuscular mycorrhiza (AM) association between roots and obligate symbiotic Glomeromycota ('AM fungi') 'feeds' about 80% of land plants. AM forming fungi supply land plants with inorganic nutrients and have an enormous impact on terrestrial ecosystems. In return, AM fungi obtain up to 20% of the plant-fixed CO(2), putatively as monosaccharides. In a recent work we have reported the characterization of the first glomeromycotan monosaccharide transporter, GpMST1, and its gene sequence. We discuss that AM fungi might take up sugars deriving from plant cell-wall material. The GpMST1 sequence delivers valuable data for the isolation of orthologues from other AM fungi and may eventually lead to the understanding of C-flows in the AM.

The Uromyces fabae UfAAT3 gene encodes a general amino acid permease that prefers uptake of in planta scarce amino acids.

SUMMARY The obligately biotrophic rust fungi are dependent on nutrient supply from their host plants. A cDNA library of infection structures of the rust fungus Uromyces fabae was used to identify a gene (UfAAT3) that encodes a protein with a high degree of sequence similarity to fungal amino acid permeases. The expression profile revealed by RT-PCR shows an up-regulation very early during rust development, with the highest level in haustoria and infected leaves. Heterologous expression of UfAAT3p in Xenopus oocytes revealed an amino acid permease energized by co-transport with protons and exhibiting a broad substrate specificity. Compared to the previously described U. fabae amino acid transporter (AAT1), which represented the highest transport activities for lysine and histidine, electrophysiological measurements with cRNA of UfAAT3-injected oocytes showed substrate preferences for leucine and the sulphur containing amino acids methionine and cysteine. The unique contribution of the amino acid permeases and their substrate affinities might be connected with the availability of amino acids in the leaf tissue. Thus, in order to compare the substrate profiles of AAT1p and UfAAT3p with the natural environment of U. fabae we analysed the amino acid concentration in the apoplastic space, in addition to that in extracts of Vicia faba leaves. The predominant free amino acids were asparagine, alanine, glutamine and glutamate. However, most amino acids were present at low concentrations (between 0.02 and 0.16 mm), including the preferred substrates of the U. fabae amino acid permeases AAT1p and UfAAT3p.