Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation.

Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg(2+)-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein. To test whether lipid modification of the S-layer glycoprotein could explain these observations, H. volcanii cells were incubated with a radiolabelled precursor of isoprene, [(3)H]mevalonic acid. In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification. In the present study we report that the H. volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis. These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e. following protein translocation across the membrane

Cloning, Expression, and Purification of Functional Sec11a and Sec11b, Type I Signal Peptidases of the Archaeon Haloferax volcanii

Across evolution, type I signal peptidases are responsible for the cleavage of secretory signal peptides from proteins following their translocation across membranes. In Archaea, type I signal peptidases combine domain-specific features with traits found in either their eukaryal or bacterial counterparts. Eukaryal and bacterial type I signal peptidases differ in terms of catalytic mechanism, pharmacological profile, and oligomeric status. In this study, genes encoding Sec11a and Sec11b, two type I signal peptidases of the halophilic archaeon Haloferax volcanii, were cloned. Although both genes are expressed in cells grown in rich medium, gene deletion approaches suggest that Sec11b, but not Sec11a, is essential. For purification purposes, tagged versions of the protein products of both genes were expressed in transformed Haloferax volcanii, with Sec11a and Sec11b being fused to a cellulose-binding domain capable of interaction with cellulose in hypersaline surroundings. By employing an in vitro signal peptidase assay designed for use with high salt concentrations such as those encountered by halophilic archaea such as Haloferax volcanii, the signal peptide-cleaving activities of both isolated membranes and purified Sec11a and Sec11b were addressed. The results show that the two enzymes differentially cleave the assay substrate, raising the possibility that the Sec11a and Sec11b serve distinct physiological functions

N-glycosylation in Haloferax volcanii: adjusting the sweetness

Long believed to be restricted to Eukarya, it is now known that cells of all three domains of life perform N-glycosylation, the covalent attachment of glycans to select target protein asparagine residues. Still, it is only in the last decade that pathways of N-glycosylation in Archaea have been delineated. In the haloarchaeon Haloferax volcanii, a series of Agl (archaeal glycosylation) proteins is responsible for the addition of an N-linked pentasaccharide to modified proteins, including the surface (S)-layer glycoprotein, the sole component of the surface layer surrounding the cell. The S-layer glycoprotein N-linked glycosylation profile changes, however, as a function of surrounding salinity. Upon growth at different salt concentrations, the S-layer glycoprotein is either decorated by the N-linked pentasaccharide introduced above or by both this pentasaccharide as well as a tetrasaccharide of distinct composition. Recent efforts have identified Agl5–Agl15 as components of a second Hfx. volcanii N-glycosylation pathway responsible for generating the tetrasaccharide attached to S-layer glycoprotein when growth occurs in 1.75 M but not 3.4 M NaCl-containing medium

Tomato ABSCISIC ACID STRESS RIPENING (ASR) Gene Family Revisited

 Tomato ABSCISIC ACID RIPENING 1 (ASR1) was the first cloned plant ASR gene. ASR orthologs were then cloned from a large number of monocot, dicot and gymnosperm plants, where they are mostly involved in response to abiotic (drought and salinity) stress and fruit ripening. The tomato genome encodes five ASR genes: ASR1, 2, 3 and 5 encode low-molecular-weight proteins (ca. 110 amino acid residues each), whereas ASR4 encodes a 297-residue polypeptide. Information on the expression of the tomato ASR gene family is scarce. We used quantitative RT-PCR to assay the expression of this gene family in plant development and in response to salt and osmotic stresses. ASR1 and ASR4 were the main expressed genes in all tested organs and conditions, whereas ASR2 and ASR3/5 expression was two to three orders of magnitude lower (with the exception of cotyledons). ASR1 is expressed in all plant tissues tested whereas ASR4 expression is limited to photosynthetic organs and stamens. Essentially, ASR1 accounted for most of ASR gene expression in roots, stems and fruits at all developmental stages, whereas ASR4 was the major gene expressed in cotyledons and young and fully developed leaves. Both ASR1 and ASR4 were expressed in flower organs, with ASR1 expression dominating in stamens and pistils, ASR4 in sepals and petals. Steady-state levels of ASR1 and ASR4 were upregulated in plant vegetative organs following exposure to salt stress, osmotic stress or the plant abiotic stress hormone abscisic acid (ABA). Tomato plants overexpressing ASR1 displayed enhanced survival rates under conditions of water stress, whereas ASR1-antisense plants displayed marginal hypersensitivity to water withholding.Fil: Golan, Ido. Ben Gurion University of the Negev; IsraelFil: Dominguez, Pia Guadalupe. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Konrad, Zvia. Ben Gurion University of the Negev; IsraelFil: Inbar, Moshe. Ben Gurion University of the Negev; IsraelFil: Carrari, Fernando Oscar. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bar-Zvi, Dudy. Ben Gurion University of the Negev; Israe

ASR1-overexpressing tomato plants have enhanced tolerance to water stress.

<p>Plants were grown in pots in the greenhouse using optimal irrigation for 17 days. Water was withheld for 22 days, following by rewatering for 17 days. Panels A and B show representative plants (left to right): wild type, <i>ASR1</i>-OE-31, <i>ASR1</i>-OE-12, <i>ASR1</i>-OE-16 after 22 days of dehydration (A) and 17 days of rewatering (B). Panel C, quantitative data of survival of three lines of <i>ASR1</i>-overexpressing (<i>ASR1</i>-OE) plants and four lines of <i>ASR1</i>-antisense (<i>ASR1</i>-AS) plants, measured at the end of the rewatering stage. Data shown are average ± SE. Bars with different letters represent statistically different values by Tukey’s HSD post-hoc test (P≤0.01).</p

Expression of the tomato <i>ASR</i> genes in flower organs.

<p>Flowers and developing leaves were harvested from greenhouse-grown tomato plants. Flowers were dissected, and steady-state levels of the indicated genes were determined. The expression levels were normalized for each of the genes to their expression in young developing leaves, defined as 1. Data shown are average ± SE. Bars with different letters represent statistically different values by Tukey’s HSD post-hoc test (P≤0.05).</p

Effects of ABA, NaCl and PEG on the steady-state transcript levels of <i>ASR</i> genes.

<p>One-week-old hydroponically grown seedlings were transferred to fresh Hoagland solution containing: no addition (white bars), 40 µM ABA (light gray bars), 0.2 M NaCl (dark gray bars), or 8% PEG 8000 (black bars). Leaves were harvested 24 h later and expression levels were determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107117#s2" target="_blank">Materials and Methods</a>. Data were normalized for each of the genes to their expression in young developing leaves, defined as 1. Data shown are average ± SE. Expression of each gene in response to plant treatment was analyzed separately using Tukey’s HSD post-hoc test. Bars with different letters represent statistically different values by (P≤0.05).</p

Expression of the <i>ASR</i> genes in fruit development.

<p>Fruits and developing leaves were harvested from greenhouse-grown tomato plants, and steady-state levels of the indicated genes were determined. The expression levels were normalized for each of the genes to their expression in young developing leaves, defined as 1. Fruit stages were defined according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107117#pone.0107117-Gillaspy1" target="_blank">[75]</a>: IG, immature green; MG, mature green; BR, breaker; OR, orange; RE, red. Data shown are average ± SE. Bars with different letters represent statistically different values by Tukey’s HSD post-hoc test (P≤0.05).</p