The gene family coding for the light-harvesting polypeptides of Photosystem I of the red alga Galdieria sulphuraria*

Recently [Marquardt et al. (2000) Gene 255: 257–265], we isolated a gene encoding a polypeptide of the light-harvesting complex of Photosystem I (LHC I) of the red alga Galdieria sulphuraria. By screening a G. sulphuraria cDNA library with a DNA probe coding for the conserved first transmembrane helix of this protein we isolated four additional genes coding for LHC I polypeptides. The deduced preproteins had calculated molecular masses of 24.6–25.6 kDa and isoelectric points of 8.09–9.82. N-terminal sequencing of a LHC I polypeptide isolated by gel electrophoresis allowed us to determine the cleavage site of the transit peptide of one of the deduced polypeptides. The mature protein has a calculated molecular mass of 20.6 kDa and an isoelectric point of 7.76. The genes were amplified from nuclear G. sulphuraria DNA by polymerase chain reaction (PCR) using oligonucleotides annealing in the regions of the start and stop codons as primers. All genomic sequences were 80–300 base pairs longer than the PCR products obtained from the respective cDNA clones, pointing to the existence of 1–5 introns per gene. The G. sulphuraria genes form a homogeneous gene family with overall pairwise amino acid identities of 46.0–56.6%. Homology to two diatom, one cryptophytic and two higher plant light-harvesting polypeptides was lower with pairwise identities of 21.1–34.1%. Only one diatom polypeptide showed a higher degree of identity of up to −39.3%. This revised version was published online in June 2006 with corrections to the Cover Date.     

The NMR solution structure of the 30S ribosomal protein S27e encoded in gene RS27_ARCFU of Archaeoglobus fulgidis reveals a novel protein fold

The Archaeoglobus fulgidis gene RS27_ARCFU encodes the 30S ribosomal protein S27e. Here, we present the high-quality NMR solution structure of this archaeal protein, which comprises a C4 zinc finger motif of the CX(2)CX(14-16)CX(2)C class. S27e was selected as a target of the Northeast Structural Genomics Consortium (target ID: GR2), and its three-dimensional structure is the first representative of a family of more than 116 homologous proteins occurring in eukaryotic and archaeal cells. As a salient feature of its molecular architecture, S27e exhibits a beta-sandwich consisting of two three-stranded sheets with topology B(decreasing), A(increasing), F(decreasing), and C(increasing), D(decreasing), E(increasing). Due to the uniqueness of the arrangement of the strands, the resulting fold was found to be novel. Residues that are highly conserved among the S27 proteins allowed identification of a structural motif of putative functional importance; a conserved hydrophobic patch may well play a pivotal role for functioning of S27 proteins, be it in archaeal or eukaryotic cells. The structure of human S27, which possesses a 26-residue amino-terminal extension when compared with the archaeal S27e, was modeled on the basis of two structural templates, S27e for the carboxy-terminal core and the amino-terminal segment of the archaeal ribosomal protein L37Ae for the extension. Remarkably, the electrostatic surface properties of archaeal and human proteins are predicted to be entirely different, pointing at either functional variations among archaeal and eukaryotic S27 proteins, or, assuming that the function remained invariant, to a concerted evolutionary change of the surface potential of proteins interacting with S27.     

Light-harvesting proteins of diatoms: Their relationship to the chlorophyll a/b binding proteins of higher plants and their mode of transport into plastids

Summary We have cloned and characterized members of a gene family encoding polypeptide constituents of the fucoxanthin, chlorophyll a/c protein complex, a light-harvesting complex associated with photosystem II of diatoms and brown algae. Three cDNA clones encoding proteins associated with this complex in the diatom Phaeodactylum tricornutum have been isolated. As deduced from the nucleotide sequences, these light-harvesting proteins show homology to the chlorophyll a/b binding polypeptides of higher plants. Specifically, the N-terminal regions of the fucoxanthin, chlorophyll a/c-binding proteins are homologous to the chlorophyll a/b binding proteins in both the third membrane-spanning domain and the stroma-exposed region between membrane-spanning domains 2 and 3. Like the chlorophyll a/b-binding proteins, the mature fucoxanthin, chlorophyll a/c polypeptides have three hydrophobic -helical domains which could span the membrane bilayer. The similarities between the two light-harvesting proteins might reflect the fact that both bind chlorophyll molecules and/or might be important for maintaining certain structural features of the complex. There is little similarity between the N-terminal sequences of the primary translation products of the fucoxanthin, chlorophyll a/c proteins and any transit sequences that have been characterized. Instead, the N-terminal sequences have features resembling those of signal sequences. Thus either transit peptides used in P. tricornutum show little resemblance to those of higher plants and green algae or the nuclear-encoded plastid proteins enter the organelle via a mechanism different from that used in higher plants.     

The Arabidopsis nuclear DAL gene encodes a chloroplast protein which is required for the maturation of the plastid ribosomal RNAs and is essential for chloroplast differentiation

Altered pigmentation is an easily scored and sensitive monitor of plastid function. We analyzed in detail a yellow colored transposon-tagged mutant (dal1-2) that is allelic to the dal mutant previously identified (Babiychuk et al., 1997). Mesophyll cells of mutant plants possess abnormal nucleoids and more but smaller plastids than wild type cells. Plastid development in dal1-2 is not altered in the dark but is arrested at the early steps of thylakoid assembly. The amino acid sequence of the protein deduced from our cDNA clone is 21 amino acids longer than the previously published DAL sequence (Babiychuk et al., 1997) and allowed us to show that DAL codes for a chloroplast protein. The dal1-2 mutation has a global negative effect on plastid RNA accumulation and on expression of nuclear encoded photosynthetic genes. We show that the plastid RNA polymerases, the nuclear-encoded NEP and the plastid-encoded PEP, are functional in the mutant. Precursor 16S and 23S rRNA species specifically accumulate at a high level in the mutant but the 5-end and the long 3-end trailer are not modified. We suggest that the dal mutation is involved in plastid rRNA processing and consequently in translation and early chloroplast differentiation.     

SecA is plastid-encoded in a red alga: implications for the evolution of plastid genomes and the thylakoid protein import apparatus

Summary Partial sequence analysis of the plastid DNA (ptDNA) from a red alga, Antithamnion sp., revealed the presence of a homologue to the Escherichia coli SecA gene as well as two open reading frames (ORF 510, ORF 179). In addition a sec Y homologue has been detected on the plastid genome by heterologous hybridization. None of these genes has been found in completely sequenced chlorophytic plastid genomes. SecA and secY gene copies were also detected in the ptDNA of a chromophytic alga, indicating that secAY may be ubiquitous in rhodophytes and chromophytes. The significance of these findings for the evolution of plastid genomes and the thylakoid protein import mechanism is discussed.     

The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein

The effect of light on the biosynthesis of the light-harvesting chlorophyll a/b protein (LHCP) is investigated in wild-type barley (Hordeum vulgare L.) and in the chlorophyll b-less mutant chlorina f2. In dark-grown plants a short red light pulse triggers the appearance of mRNA activity for the LHCP. While the accumulation of this mRNA is controlled by phytochrome (Apel (1979) Eur. J. Biochem. 97, 183–188), the red light treatment is not sufficient to induce the appearance of the LHCP within the membrane. Thus, at least one of the subsequent steps in the biosynthetic pathway leading to the assembly of the LHCP is controlled by light. The red light-induced mRNA is taken up into the polysomes during the subsequent dark period and is translated in vitro in a cell-free protein synthesizing system. However, an accumulation of the freshly synthesized polypeptide within the plant is not observed. The apparent instability of the polypeptide might be explained by the deficiency of chlorophyll in the red light-treated plants. In the chlorophyll b-less barley mutant chlorina f2 an accumulation of the freshly synthesized apoprotein of the LHCP can be observed in the light. Thus, chlorophyll a formation seems to be a light-dependent step which is required for the stabilization of the LHCP.Abbreviations mRNA messenger RNA - EDTA ethylenediaminetetraacetic acid - SDS sodium dodecylsulfate - LHCP light-harvesting chlorophyll a/b protein     

Cloning and expression of a gene coding for the major light-harvesting chlorophyll a/b protein of photosystem II in the green alga <Emphasis Type="Italic">Dunaliella salina</Emphasis>

Genes encoding proteins of the major light-harvesting complex of photosystem II (LHCII) in higher plants are well studied. However, little is known about the corresponding genes in the green alga Dunaliella salina, although this knowledge might provide valuable information about the respective roles of each LHCII protein at the molecular level under extreme environmental conditions. Here, we describe an additional LhcII gene from D. salina. An LhcII cDNA cloned by screening a D. salina cDNA library contains an open reading frame encoding a protein of 261 amino acids with a calculated molecular mass of 27.8 kDa. The deduced amino acid sequence shows high homology with other LHCII proteins. Genomic DNA—obtained by PCR using a specific primer set corresponding to the 5′ and 3′ untranslated regions—was used to determine the intron-exon structure. Short-term changes in mRNA levels after a shift from low-light to high-light or dark conditions were analyzed by real-time quantitative PCR, and indicated that this gene expresses different mRNA levels under different light conditions.     

Autophagy-related gene 7 is downstream of heat shock protein 27 in the regulation of eye morphology,polyglutamine toxicity,and lifespan in Drosophila

Background

Autophagy and molecular chaperones both regulate protein homeostasis and maintain important physiological functions. Atg7 (autophagy-related gene 7) and Hsp27 (heat shock protein 27) are involved in the regulation of neurodegeneration and aging. However, the genetic connection between Atg7 and Hsp27 is not known.

Methods

The appearances of the fly eyes from the different genetic interactions with or without polyglutamine toxicity were examined by light microscopy and scanning electronic microscopy. Immunofluorescence was used to check the effect of Atg7 and Hsp27 knockdown on the formation of autophagosomes. The lifespan of altered expression of Hsp27 or Atg7 and that of the combination of the two different gene expression were measured.

Results

We used the Drosophila eye as a model system to examine the epistatic relationship between Hsp27 and Atg7. We found that both genes are involved in normal eye development, and that overexpression of Atg7 could eliminate the need for Hsp27 but Hsp27 could not rescue Atg7 deficient phenotypes. Using a polyglutamine toxicity assay (41Q) to model neurodegeneration, we showed that both Atg7 and Hsp27 can suppress weak, toxic effect by 41Q, and that overexpression of Atg7 improves the worsened mosaic eyes by the knockdown of Hsp27 under 41Q. We also showed that overexpression of Atg7 extends lifespan and the knockdown of Atg7 or Hsp27 by RNAi reduces lifespan. RNAi-knockdown of Atg7 expression can block the extended lifespan phenotype by Hsp27 overexpression, and overexpression of Atg7 can extend lifespan even under Hsp27 knockdown by RNAi.

Conclusions

We propose that Atg7 acts downstream of Hsp27 in the regulation of eye morphology, polyglutamine toxicity, and lifespan in Drosophila.     

Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.     

The effect of NaCl on water relations,chlorophyll, and protein and proline contents of two cultivars of blackgram (Vigna mungo L.)

The physiological basis of salt tolerance of two cultivars of blackgram, cv Candhari Mash (relatively salt tolerant) and cv Mash 654 (salt sensitive), was assessed in salinized sand culture at the flowering stage. Increasing NaCl concentration in the rooting medium significantly reduced the chlorophyll a, chlorophyll b, and total chlorophyll, leaf water potential (Ψ_w), leaf solute potential (Ψ_s), and leaf turgor potential (Ψ_p) in both the cultivars. Leaf protein and proline content was increased as a result of increasing salt concentration in both cultivars. High salt concentrations had no significant effect on the seed protein content of both cultivars. At high salinities, cv Candhari Mash had significantly greater chlorophyll a, chlorophyll b and total chlorophyll, leaf water potential, solute potential, and turgor potential than cv Mash 654, but the latter had greater leaf proline content than cv Candhari Mash. Cultivars did not differ significantly for both leaf and seed protein contents. The relatively salt tolerant cv Candhari Mash maintained high leaf water potential and turgor potential to resist salt injury. Leaf proline content had negative correlation with salt tolerance in blackgram.     

The complete mitochondrial genome of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae: gene order and trn gene clusters reveal a common evolutionary course for all Sordariomycetes, while intergenic regions show variation

The mitochondrial genome (mtDNA) of the entomopathogenic fungus Metarhizium anisopliae var. anisopliae, with a total size of 24,673 bp, was one of the smallest known mtDNAs of Pezizomycotina. It contained the 14 typical genes coding for proteins related to oxidative phosphorylation, the two rRNA genes, a single intron that harbored an intronic ORF coding for a putative ribosomal protein (rps) within the large rRNA gene (rnl), and a set of 24 tRNA genes which recognized codons for all amino acids, except proline and valine. Gene order comparison with all known mtDNAs of Sordariomycetes illustrated a highly conserved genome organization for all the protein- and rRNA-coding genes, as well as three clusters of tRNA genes. By considering all mitochondrial essential protein-coding genes as one unit a phylogenetic study of these small genomes strongly supported the common evolutionary course of Sordariomycetes (100% bootstrap support) and highlighted the advantages of analyzing small genomes (mtDNA) over single genes. In addition, comparative analysis of three intergenic regions demonstrated sequence variability that can be exploited for intra- and inter-specific identification of Metarhizium. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

The fadD gene of Escherichia coli K12 is located close to rnd at 39.6 min of the chromosomal -map and is a new member of the AMP-binding protein family

The fadD gene of Escherichia coli K12 was cloned and sequenced. The gene was identified by its ability to complement the corresponding mutant and by measuring the enzymatic activity after its expression in this mutant. The deduced polypeptide sequence exhibits similarity to other long chain acyl-CoA (coenzyme A) synthetases and a variety of other proteins, which together form a family of AMP-binding proteins. This family is extended by several new members and subdivided into four groups. fadD is assigned to a subgroup that does not include long chain acyl-CoA synthetases from eukaryotic organisms.