Identification and structural characterization of FYVE domain-containing proteins of Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>FYVE domains have emerged as membrane-targeting domains highly specific for phosphatidylinositol 3-phosphate (PtdIns(3)<it>P</it>). They are predominantly found in proteins involved in various trafficking pathways. Although FYVE domains may function as individual modules, dimers or in partnership with other proteins, structurally, all FYVE domains share a fold comprising two small characteristic double-stranded β-sheets, and a C-terminal α-helix, which houses eight conserved Zn²⁺ion-binding cysteines. To date, the structural, biochemical, and biophysical mechanisms for subcellular targeting of FYVE domains for proteins from various model organisms have been worked out but plant FYVE domains remain noticeably under-investigated.</p> <p>Results</p> <p>We carried out an extensive examination of all <it>Arabidopsis </it>FYVE domains, including their identification, classification, molecular modeling and biophysical characterization using computational approaches. Our classification of fifteen <it>Arabidopsis </it>FYVE proteins at the outset reveals unique domain architectures for FYVE containing proteins, which are not paralleled in other organisms. Detailed sequence analysis and biophysical characterization of the structural models are used to predict membrane interaction mechanisms previously described for other FYVE domains and their subtle variations as well as novel mechanisms that seem to be specific to plants.</p> <p>Conclusions</p> <p>Our study contributes to the understanding of the molecular basis of FYVE-based membrane targeting in plants on a genomic scale. The results show that FYVE domain containing proteins in plants have evolved to incorporate significant differences from those in other organisms implying that they play a unique role in plant signaling pathways and/or play similar/parallel roles in signaling to other organisms but use different protein players/signaling mechanisms.</p

A Novel Insight into the Oxidoreductase Activity of Helicobacter pylori HP0231 Protein.

Altogether our results show that HP0231 is an oxidoreductase that catalyzes disulfide bond formation in the periplasm. We propose to call it HpDsbA

Redox equilibrium of <i>H. pylori</i> HP0231 with glutathione.

<p>The fraction of reduced (R) HP0231 was determined using the specific HP0231 fluorescence at 324 nm.</p

Redox state of HP0231 in WT, mutants: <i>dsbI</i>::<i>aph, hp0231</i>::<i>cat</i> and complemented strains.

<p>Bacterial cultures were treated with 10% TCA, followed by alkylation with AMS. Cellular proteins including the reduced (red; DTT treated, modified with AMS) and the oxidized (ox; non-modified with AMS) controls were separated by 18% SDS-PAGE under non-reducing conditions, and Western blot analysis using antibodies against HP0231 was performed. Each lane contains proteins isolated from the same amount of bacteria.</p

Motility of <i>H. pylori</i> N6 strains: wt, <i>hp0231::cat</i> and <i>hp0231::cat</i> complemented <i>in trans</i> by puWM397 (<i>hp0231⁺</i>).

<p>Bacterial motility was monitored after 4 days of incubation on 0.3% MH-agar plates containing 10% FCS. The <i>hp0231</i> mutant strain is non-motile.</p

Primers used in this study.

<p>Restriction recognition sites introduced for cloning purposes are underlined. All primers were designed on the basis of <i>H. pylori</i> 26695 genome nucleotide sequence.</p

Bacterial strains and plasmids used in this study.

<p>Bacterial strains and plasmids used in this study.</p