Noam Adir

Photoinhibition — a historical perspective

Photoinhibition is a state of physiological stress that occurs in all oxygen evolving photosynthetic organisms exposed to light. The primary damage occurs within the reaction center of Photosystem II (PS II). While irreversible photoinduced damage to PS II occurs at all light intensities, the efficiency of photosynthetic electron transfer decreases markedly only when the rate of damage exceeds the rate of its repair, which requires de novo PS II protein synthesis. Photoinhibition has been studied for over a century using a large variety of biochemical, biophysical and genetic methodologies. The discovery of the light induced turnover of a protein, encoded by the plastid psbA gene (the D1 protein), later identified as one of the photochemical reaction center II proteins, has led to the elucidation of the underlying mechanism of photoinhibition and to a deeper understanding of the PS II ‘life cycle.’

Elucidation of the molecular structures of components of the phycobilisome: reconstructing a giant

The molecular architectures of photosynthetic complexes are rapidly becoming available through the power of X-ray crystallography. These complexes are comprised of antenna complexes, which absorb and transfer energy into photochemical reaction centers. Most reaction centers, found in both oxygenic and non-oxygenic species, are connected to transmembrane chlorophyll containing antennas, and the crystal structures of these antennas contain information on the structure of the entire complex as well as clear indications on their modes of functional association. In cyanobacteria and red alga, most of the Photosystem II associated light harvesting is performed by an enormous (3–7 MDa) membrane attached complex called the phycobilisome (PBS). While the crystal structures of many isolated components of different PBSs have been determined, the structure of the entire complex as well as its manner of association with Photosystem II can only be suggested. In this review, the structural information obtained on the isolated components will be described. The structural information obtained from the components provides the basis for the modeled reconstruction of this giant complex.

Transcriptional regulation of meiosis in budding yeast.

Initiation of meiosis in Saccharomyces cerevisiae is regulated by mating type and nutritional conditions that restrict meiosis to diploid cells grown under starvation conditions. Specifically, meiosis occurs in MATa/MATalpha cells shifted to nitrogen depletion media in the absence of glucose and the presence of a nonfermentable carbon source. These conditions lead to the expression and activation of Ime 1, the master regulator of meiosis. IME1 encodes a transcriptional activator recruited to promoters of early meiosis-specific genes by association with the DNA-binding protein, Ume6. Under vegetative growth conditions these genes are silent due to recruitment of the Sin3/Rpd3 histone deacetylase and Isw2 chromatin remodeling complexes by Ume6. Transcription of these meiotic genes occurs following histone acetylation by Gcn5. Expression of the early genes promote entry into the meiotic cycle, as they include genes required for premeiotic DNA synthesis, synapsis of homologous chromosomes, and meiotic recombination. Two of the early meiosis specific genes, a transcriptional activator, Ndt80, and a CDK2 homologue, Ime2, are required for the transcription of middle meiosis-specific genes that are involved with nuclear division and spore formation. Spore maturation depends on late genes whose expression is indirectly dependent on Ime1, Ime2, and Ndt80. Finally, phosphorylation of Imel by Ime2 leads to its degradation, and consequently to shutting down of the meiotic transcriptional cascade. This review is focusing on the regulation of gene expression governing initiation and progression through meiosis.

Viral photosynthetic reaction center genes and transcripts in the marine environment

Cyanobacteria of the genera Synechococcus and Prochlorococcus are important contributors to photosynthetic productivity in the open ocean. The discovery of genes (psbA, psbD) that encode key photosystem II proteins (D1, D2) in the genomes of phages that infect these cyanobacteria suggests new paradigms for the regulation, function and evolution of photosynthesis in the vast pelagic ecosystem. Reports on the prevalence and expression of phage photosynthesis genes, and evolutionary data showing a potential recombination of phage and host genes, suggest a model in which phage photosynthesis genes help support photosynthetic activity in their hosts during the infection process. Here, using metagenomic data in natural ocean samples, we show that about 60% of the psbA genes in surface water along the global ocean sampling transect are of phage origin, and that the phage genes are undergoing an independent selection for distinct D1 proteins. Furthermore, we show that different viral psbA genes are expressed in the environment.

Domain Analysis of the Chloroplast Polynucleotide Phosphorylase Reveals Discrete Functions in RNA Degradation, Polyadenylation, and Sequence Homology with Exosome Proteins

The molecular mechanism of mRNA degradation in the chloroplast consists of sequential events, including endonucleolytic cleavage, the addition of poly(A)-rich sequences to the endonucleolytic cleavage products, and exonucleolytic degradation. In spinach chloroplasts, the latter two steps of polyadenylation and exonucleolytic degradation are performed by the same phosphorolytic and processive enzyme, polynucleotide phosphorylase (PNPase). An analysis of its amino acid sequence shows that the protein is composed of two core domains related to RNase PH, two RNA binding domains (KH and S1), and an α-helical domain. The amino acid sequence and domain structure is largely conserved between bacteria and organelles. To define the molecular mechanism that controls the two opposite activities of this protein in the chloroplast, the ribonuclease, polymerase, and RNA binding properties of each domain were analyzed. The first core domain, which was predicted to be inactive in the bacterial enzymes, was active in RNA degradation but not in polymerization. Surprisingly, the second core domain was found to be active in degrading polyadenylated RNA only, suggesting that nonpolyadenylated molecules can be degraded only if tails are added, apparently by the same protein. The poly(A) high-binding-affinity site was localized to the S1 domain. The complete spinach chloroplast PNPase, as well as versions containing the core domains, complemented the cold sensitivity of an Escherichia coli PNPase-less mutant. Phylogenetic analyses of the two core domains showed that the two domains separated very early, resulting in the evolution of the bacterial and organelle PNPases and the exosome proteins found in eukaryotes and some archaea.

Two newly identified membrane-associated and plastidic tomato HXKs : characteristics, predicted structure and intracellular localization

Two new tomato hexokinase genes, LeHXK3 and LeHXK4, were cloned and characterized, placing tomato as the first plant with four characterized HXK genes. Based on their sequence, LeHXK3 is the third membrane-associated (type-B) and LeHXK4 is the first plastidic (type-A) HXK identified in tomato. Expression of HXK-GFP fusion proteins in protoplasts indicated that the LeHxk3 enzyme is associated with the mitochondria while LeHxk4 is localized in plastids. Furthermore, LeHxk4::GFP fusion protein is found within stromules, suggesting transport of LeHxk4 between plastids. Structure prediction of the various plant HXK enzymes suggests that unlike the plastidic HXKs, the predicted membrane-associated HXKs are positively charged near their putative N-terminal membrane anchor domain, which might enhance their association with the negatively charged membranes. LeHxk3 and LeHxk4 were analyzed following expression in yeast. Both enzymes have higher affinity for glucose relative to fructose and are inhibited by ADP. Yet, unlike the other HXKs, the stromal HXK has higher Vmax with glucose than with fructose. Expression analysis of the four HXK genes in tomato tissues demonstrated that LeHXK1 and LeHXK4 are the dominant HXKs in all tissues examined. Notably, the plastidic LeHXK4 is expressed in all tissues including starchless, non-photosynthetic sink tissues, such as pink and red fruits, implying phosphorylation of imported hexoses in plastids. It has been suggested that trehalose 6-phosphate (T6P) might inhibit HXK activity. However, none of the yeast-expressed tomato HXK genes was sensitive either to T6P or to trehalose, suggesting that unlike fungi HXKs, plant HXKs are not regulated by T6P.

The Proteasome and the Delicate Balance between Destruction and Rescue

The proteasome is a large multiprotein complex that degrades unwanted cellular proteins. The mechanisms that control this protein-eating machine are being uncovered

Alopecia, Neurological Defects, and Endocrinopathy Syndrome Caused by Decreased Expression of RBM28, a Nucleolar Protein Associated with Ribosome Biogenesis

Single-gene disorders offer unique opportunities to shed light upon fundamental physiological processes in humans. We investigated an autosomal-recessive phenotype characterized by alopecia, progressive neurological defects, and endocrinopathy (ANE syndrome). By using homozygosity mapping and candidate-gene analysis, we identified a loss-of-function mutation in RBM28, encoding a nucleolar protein. RBM28 yeast ortholog, Nop4p, was previously found to regulate ribosome biogenesis. Accordingly, electron microscopy revealed marked ribosome depletion and structural abnormalities of the rough endoplasmic reticulum in patient cells, ascribing ANE syndrome to the restricted group of inherited disorders associated with ribosomal dysfunction.

Allophycocyanin Trimer Stability and Functionality Are Primarily Due to Polar Enhanced Hydrophobicity of the Phycocyanobilin Binding Pocket

Allophycocyanin (APC) is the primary pigment-protein component of the cores of the phycobilisome antenna complex. In addition to an extremely high degree of amino acid sequence conservation, the overall structures of APC from both mesophilic and thermophilic species are almost identical at all levels of assembly, yet APC from thermophilic organisms should have structural attributes that prevent thermally induced denaturation. We determined the structure of APC from the thermophilic cyanobacterium Thermosynechococcus vulcanus to 2.9 A, reaffirming the conservation of structural similarity with APC from mesophiles. We provide spectroscopic evidence that T. vulcanus APC is indeed more stable at elevated temperatures in vitro, when compared with the APC from mesophilic species. APC thermal and chemical stability levels are further enhanced when monitored in the presence of high concentrations of buffered phosphate, which increases the strength of hydrophobic interactions, and may mimic the effect of cytosolic crowding. Absorption spectroscopy, size-exclusion HPLC, and native gel electrophoresis also show that the thermally or chemically induced changes in the APC absorption spectra that result in the loss of the prominent 652-nm band in trimeric APC are not a result of physical monomerization. We propose that the bathochromic shift that occurs in APC upon trimerization is due to the coupling of the hydrophobicity of the alpha84 phycocyanobilin cofactor environment created by a deep cleft formed by the beta subunit with highly charged flanking regions. This arrangement also provides the additional stability required by thermophiles at elevated temperatures. The chemical environment that induces the bathochromic shift in APC trimers is different from the source of shifts in the absorption of monomers of the terminal energy acceptors APC(B) and L(CM), as visualized by the building of molecular models.

Allophycocyanin and phycocyanin crystal structures reveal facets of phycobilisome assembly.

X-ray crystal structures of the isolated phycobiliprotein components of the phycobilisome have provided high resolution details to the description of this light harvesting complex at different levels of complexity and detail. The linker-independent assembly of trimers into hexamers in crystal lattices of previously determined structures has been observed in almost all of the phycocyanin (PC) and allophycocyanin (APC) structures available in the Protein Data Bank. In this paper we describe the X-ray crystal structures of PC and APC from Synechococcus elongatus sp. PCC 7942, PC from Synechocystis sp. PCC 6803 and PC from Thermosynechococcus vulcanus crystallized in the presence of urea. All five structures are highly similar to other PC and APC structures on the levels of subunits, monomers and trimers. The Synechococcus APC forms a unique loose hexamer that may show the structural requirements for core assembly and rod attachment. While the Synechococcus PC assembles into the canonical hexamer, it does not further assemble into rods. Unlike most PC structures, the Synechocystis PC fails to form hexamers. Addition of low concentrations of urea to T. vulcanus PC inhibits this proteins propensity to form hexamers, resulting in a crystal lattice composed of trimers. The molecular source of these differences in assembly and their relevance to the phycobilisome structure is discussed.