Interfering RNA was used to suppress the expression of two genes that encode the manganese-stabilizing protein of photosystem II in Arabidopsis thaliana, MSP-1 (encoded by psbO-1, At5g66570), and MSP-2 (encoded by psbO-2, At3g50820). A phenotypic series of transgenic plants was recovered that expressed high, intermediate, and low amounts of these two manganese-stabilizing proteins. Chlorophyll fluorescence induction and decay analyses were performed. Decreasing amounts of expressed protein led to the progressive loss of variable fluorescence and a marked decrease in the fluorescence quantum yield (F v /F m ) in both the absence and the presence of dichloromethylurea. This result indicated that the amount of functional photosystem II reaction centers was compromised in the plants that exhibited intermediate and low amounts of the manganese-stabilizing proteins. An analysis of the decay of the variable fluorescence in the presence of dichlorophenyldimethylurea indicated that charge recombination between Q A ؊ and the S 2 state of the oxygen-evolving complex was seriously retarded in the plants that expressed low amounts of the manganesestabilizing proteins. This may have indicated a stabilization of the S 2 state in the absence of the extrinsic component. Immunological analysis of the photosystem II protein complement indicated that significant losses of the CP47, CP43, and D1 proteins occurred upon the loss of the manganese-stabilizing proteins. This indicated that these extrinsic proteins were required for photosystem II core assembly/stability. Additionally, although the quantity of the 24-kDa extrinsic protein was only modestly affected by the loss of the manganese-stabilizing proteins, the 17-kDa extrinsic protein dramatically decreased. The control proteins ribulose bisphosphate carboxylase and cytochrome f were not affected by the loss of the manganese-stabilizing proteins; the photosystem I PsaB protein, however, was significantly reduced in the low expressing transgenic plants. Finally, it was determined that the transgenic plants that expressed low amounts of the manganese-stabilizing proteins could not grow photoautotrophically.In higher plants and cyanobacteria, at least six intrinsic proteins appear to be required for oxygen evolution by photosystem II (PS II) 1 (1-3). These are CP 47, CP 43, the D1 and D2proteins, and the ␣ and ␤ subunits of cytochrome b 559 . Insertional inactivation or deletion of the genes for these components results in the complete loss of oxygen evolution activity. Additionally, a number of low molecular mass, intrinsic membrane protein components are associated with PS II (4 -6), although the functions of many of these proteins remain obscure. Although PS II complexes containing only these intrinsic components can evolve oxygen, they do so at low rates (about 25-40% of control), are extremely susceptible to photoinactivation, and require high, non-physiological levels of calcium and chloride for maximal activity (1, 3). In higher plants, three extrinsic proteins, with apparent...

Acetyl-CoA carboxylase is a biotin-dependent enzyme that catalyzes the regulated step in fatty acid synthesis. The bacterial form has three separate components: biotin carboxylase, biotin carboxyl carrier protein (BCCP), and carboxyltransferase. Catalysis by acetyl-CoA carboxylase proceeds via two half-reactions. In the first half-reaction, biotin carboxylase catalyzes the ATP-dependent carboxylation of biotin, which is covalently attached to BCCP, to form carboxybiotin. In the second half-reaction, carboxyltransferase transfers the carboxyl group from carboxybiotin to acetyl-CoA to form malonyl-CoA. All biotin-dependent carboxylases are proposed to have a two-site ping-pong mechanism in which the carboxylase and transferase activities are separate and do not interact. This posits two hypotheses: either biotin carboxylase and BCCP undergo the first half-reaction, BCCP dissociates, and then BCCP binds to carboxyltransferase, or all three constituents form an enzyme complex. To determine which hypothesis is correct, a steady-state enzyme kinetic analysis of Escherichia coli acetyl-CoA carboxylase was conducted. The results indicated the two active sites of acetyl-CoA carboxylase interact. Both in vitro and in vivo pull-down assays demonstrated that the three components of E. coli acetyl-CoA carboxylase form a multimeric complex and that complex formation is unaffected by acetyl-CoA, AMPPNP, and mRNA encoding carboxyltransferase. The implications of these findings for the regulation of acetyl-CoA carboxylase and fatty acid biosynthesis are discussed.

When mammalian mitochondria are exposed to high calcium and phosphate, a massive swelling, uncoupling of respiration, and release of cytochrome c occur. These changes are mediated by opening of the mitochondrial permeability transition pore (MPTP). Activation of the MPTP in vivo in response to hypoxic and oxidative stress leads to necrotic and apoptotic cell death. Considering that embryos of the brine shrimp Artemia franciscana tolerate anoxia for years, we investigated the MPTP in this crustacean to reveal whether pore opening occurs. Minimum molecular constituents of the regulated MPTP in mammals are believed to be the voltage-dependent anion channel, the adenine nucleotide translocators, and cyclophilin D. Western blot analysis revealed that mitochondria from A. franciscana possess all three required components. When measured with a calcium-sensitive fluorescent probe, rat liver mitochondria are shown to release matrix calcium after addition of ≥100 μM extramitochondrial calcium (MPTP opening), whereas brine shrimp mitochondria continue to take up extramitochondrial calcium and do not release internal stores even up to 1.0 mM exogenously added calcium (no MPTP opening). Furthermore, no swelling of A. franciscana mitochondria in response to added calcium was observed, and no release of cytochrome c could be detected. HgCl2-dependent swelling and cytochrome c release were readily confirmed, which is consistent with the presence of an “unregulated pore.” Although the absence of a regulated MPTP in A. franciscana mitochondria could contribute to the extreme hypoxia tolerance in this species, we speculate that absence of the regulated MPTP may be a general feature of invertebrates.

A mutation was recovered in the slr0721 gene, which encodes the decarboxylating NADP ؉ -dependent malic enzyme in the cyanobacterium Synechocystis sp. strain PCC 6803, yielding the mutant 3WEZ. Under continuous light, 3WEZ exhibits poor photoautotrophic growth while growing photoheterotrophically on glucose at rates nearly indistinguishable from wild-type rates. Interestingly, under diurnal light conditions (12 h of light and 12 h of dark), normal photoautotrophic growth of the mutant is completely restored.Cyanobacteria are photoautotrophic gram-negative eubacteria that are capable of performing oxygenic photosynthesis. Synechocystis sp. strain PCC 6803 is a naturally transformable (7) unicellular cyanobacterium and has proven to be one of the best model organisms for studying the mechanism and regulation of oxygenic photosynthesis (14); it has also been used in a variety of global gene expression (6,8,13) and metabolomic (15, 16) studies. In addition to autotrophic growth, the presence of an unidentified mutation in the Williams strain of Synechocystis (14) confers glucose tolerance to this organism. With glucose as a carbon source, this strain can be grown under mixotrophic, photoheterotrophic (continuous photosynthetic illumination at 20 to 40 mol of photons m Ϫ2 s Ϫ1 in the presence of the photosystem II inhibitor dichloromethylurea [DCMU]), and heterotrophic (nonphotosynthetic continuous illumination at Ͻ1 mol of photons m Ϫ2 s Ϫ1

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