Feedback mechanisms that dissipate excess photoexcitations in light-harvesting complexes (LHCs) are necessary to avoid detrimental oxidative stress in most photosynthetic eukaryotes. Here we demonstrate the unique ability of LHCSR, a stress-related LHC from the model organism Chlamydomonas reinhardtii, to sense pH variations, reversibly tuning its conformation from a light-harvesting state to a dissipative one. This conformational change is induced exclusively by the acidification of the environment, and the magnitude of quenching is correlated to the degree of acidification of the environment. We show that this ability to respond to different pH values is missing in the related major LHCII, despite high structural homology. Via mutagenesis and spectroscopic characterization, we show that LHCSR's uniqueness relies on its peculiar C-terminus subdomain, which acts as a sensor of the lumenal pH, able to tune the quenching level of the complex.

In mammalian cells, the repair of DNA bases that have been damaged by reactive oxygen species is primarily initiated by a series of DNA glycosylases that include OGG1, NTH1, NEIL1, and NEIL2. To explore the functional significance of NEIL1, we recently reported that neil1 knock-out and heterozygotic mice develop the majority of symptoms of metabolic syndrome (Vartanian, V., Lowell, B., Minko, I. G., Wood, T. G., Ceci, J. D., George, S., Ballinger, S. W., Corless, C. L., McCullough, A. K., and Lloyd, R. S. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1864 -1869). To determine whether this phenotype could be causally related to human disease susceptibility, we have characterized four polymorphic variants of human NEIL1. Although three of the variants (S82C, G83D, and D252N) retained near wild type levels of nicking activity on abasic (AP) site-containing DNA, G83D did not catalyze the wild type ␤,␦-elimination reaction but primarily yielded the ␤-elimination product. The AP nicking activity of the C136R variant was significantly reduced. Glycosylase nicking activities were measured on both thymine glycol-containing oligonucleotides and ␥-irradiated genomic DNA using gas chromatography/mass spectrometry. Two of the polymorphic variants (S82C and D252N) showed near wild type enzyme specificity and kinetics, whereas G83D was devoid of glycosylase activity. Although insufficient quantities of C136R could be obtained to carry out gas chromatography/mass spectrometry analyses, this variant was also devoid of the ability to incise thymine glycol-containing oligonucleotide, suggesting that it may also be glycosylase-deficient. Extrapolation of these data suggests that individuals who are heterozygous for these inactive variant neil1 alleles may be at increased risk for metabolic syndrome.A major source of DNA lesions in eukaryotic cells is the interaction of reactive oxygen species with DNA constituents.DNA bases are particularly susceptible to reactive oxygen species, with the major DNA damages being, among others, 8-hydroxyguanine (8-OH-Gua), 3 8-hydroxyadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), and 4,6-diamino-5-formamido pyrimidine (FapyAde) (1, 2). To reverse the potentially deleterious effects of oxidatively induced DNA base lesions, cells primarily utilize the base excision repair pathway to restore the DNA to its original state. This pathway is initiated by lesion-specific DNA glycosylases that hydrolyze the bond attaching the damaged base to the deoxyribose, and many of these enzymes also possess an activity that catalyzes a ␤-or ␤,␦-elimination reaction at the newly formed abasic (AP) site. These incision intermediates are further processed by an AP endonuclease to yield a free 3Ј-OH that serves as a primer for repair synthesis and ligation. To initiate repair of oxidatively induced DNA base lesions, mammalian cells primarily use the products of the ogg1, nth1, neil1, and neil2 genes (reviewed in Ref.2).Human and mouse NEIL1 proteins have been shown to possess a strong substrate preference fo...

The light-harvesting complexes (LHCs) of plants can regulate the energy flux to the reaction centers in response to fluctuating light by virtue of their vast conformational landscape. They do so by switching from a light-harvesting state to a quenched state, dissipating the excess absorbed energy as heat. However, isolated LHCs are prevalently in their light-harvesting state, which makes the identification of their photoprotective mechanism extremely challenging. Here, ensemble time-resolved fluorescence experiments show that monomeric CP29, a minor LHC of plants, exists in various emissive states with identical spectra but different lifetimes. The photoprotective mechanism active in a subpopulation of strongly quenched complexes is further investigated via ultrafast transient absorption spectroscopy, kinetic modeling, and mutational analysis. We demonstrate that the observed quenching is due to excitation energy transfer from chlorophylls to a dark state of the centrally bound lutein.

To avoid photodamage, photosynthetic organisms are able to thermally dissipate the energy absorbed in excess in a process known as nonphotochemical quenching (NPQ). Although NPQ has been studied extensively, the major players and the mechanism of quenching remain debated. This is a result of the difficulty in extracting molecular information from in vivo experiments and the absence of a validation system for in vitro experiments. Here, we have created a minimal cell of the green alga Chlamydomonas reinhardtii that is able to undergo NPQ. We show that LHCII, the main light harvesting complex of algae, cannot switch to a quenched conformation in response to pH changes by itself. Instead, a small amount of the protein LHCSR1 (light-harvesting complex stress related 1) is able to induce a large, fast, and reversible pH-dependent quenching in an LHCII-containing membrane. These results strongly suggest that LHCSR1 acts as pH sensor and that it modulates the excited state lifetimes of a large array of LHCII, also explaining the NPQ observed in the LHCSR3-less mutant. The possible quenching mechanisms are discussed.photosynthesis | light-harvesting | nonphotochemical quenching | green algae | thylakoid membranes P hotosynthetic organisms get their energy from light and have developed a series of mechanisms to respond to the changes in light intensity that occur in their natural environment (1, 2). This is particularly important in high-light conditions, as the energy absorbed in excess can induce photodamage, eventually leading to the death of the organism. Photosynthetic organisms are equipped with many pigment-protein complexes, most of which in plants and green algae are members of the light-harvesting complex (Lhc) multigenic family (3). These complexes maximize light absorption in low light, but can easily become overexcited in high light (4), when a large part of the absorbed light cannot be used for charge separation in the reaction centers of the photosystems. Especially when the changes in light intensity are very fast, and thus protein degradation is not an option, photoprotective mechanisms need to be switched on to avoid the formation of singlet oxygen. The most rapid response to high light intensity is the dissipation of a large part of the absorbed energy as heat in a series of processes known as nonphotochemical quenching (NPQ) (1,5,6).The general idea is that the LHCs can switch between a lightharvesting conformation, characterized by a long excited-state lifetime, and a quenched (Q) conformation that shows a shorter lifetime because of the presence of competing de-excitation processes (7). How this switch is induced and the nature of these de-excitation processes is a matter of debate. It is known that NPQ is triggered by low luminal pH, which is a signal for the overexcitation of the membrane; this activates the quenching processes (5, 8), which involves the proteins PsbS (in plants and mosses) (9, 10) and/or lightharvesting complex stress related (LHCSR) (in green algae, mosses, and diatoms) (10-12)....

Results of in vitro and genetic studies have provided evidence for four pathways by which proteins are targeted to the chloroplast thylakoid membrane. Although these pathways are initially engaged by distinct substrates and involve some distinct components, an unresolved issue has been whether multiple pathways converge on a common translocation pore in the membrane. A homologue of eubacterial SecY called cpSecY is localized to the thylakoid membrane. Since SecY is a component of a protein-translocating pore in bacteria, cpSecY likely plays an analogous role. To explore the role of cpSecY, we obtained maize mutants with transposon insertions in the corresponding gene. Null cpSecY mutants exhibit a severe loss of thylakoid membrane, differing in this regard from mutants lacking cpSecA. Therefore, cpSecY function is not limited to a translocation step downstream of cpSecA. The phenotype of cpSecY mutants is also much more pleiotropic than that of double mutants in which both the cpSecA- and ΔpH-dependent thylakoid-targeting pathways are disrupted. Therefore, cpSecY function is likely to extend beyond any role it might play in these targeting pathways. CpSecY mutants also exhibit a defect in chloroplast translation, revealing a link between chloroplast membrane biogenesis and chloroplast gene expression.