Inhibition of photophosphorylation by kaempferol

Kaempferol, a naturally occurring flavonol, inhibited coupled electron transport and both cyclic and noncyclic photophosphorylation in isolated pea (Pisum sativum) chloroplasts. Over a concentration range which gave marked inhibition of ATP synthesis, there was no effect on basal or uncoupled electron flow or light-induced proton accumulation by isolated thylakoids. It is suggested that kaempferol acts as an energy transfer inhibitor.     

A COMPARISON OF CHLOROPLAST MEMBRANE SURFACES VISUALIZED BY FREEZE-ETCH AND NEGATIVE STAINING TECHNIQUES; AND ULTRASTRUCTURAL CHARACTERIZATION OF MEMBRANE FRACTIONS OBTAINED FROM DIGITONIN-TREATED SPINACH CHLOROPLASTS

Spinach chloroplast lamellae were washed free of negatively staining surface particles (carboxydismutase and coupling factor protein) and the resulting smooth-surfaced lamellae still showed the usual large (175 A) and small (110 A) particles seen by freeze-etching. Therefore, the freeze-fracture plane probably occurs along an internal surface of the chloroplast membrane. Fractions obtained by differential centrifugation of digitonin-treated chloroplast membranes were studied by negative staining, thin sectioning, and freeze-etching techniques for electron microscopy. The material sedimenting between 1,000 g and 10,000 g, enriched in photosystem II activity, was shown to consist of membrane fragments. These freeze-etched membrane fragments were found to have large particles on most of the exposed fracture faces. The large particles had the same size and distribution pattern as the 175 A particles seen in intact chloroplast membranes. The material sedimenting between 50,000 g and 144,000 g, which had only photosystem I activity, was found to consist of particles in various degrees of aggregation. Freeze-etching of this fraction revealed only small particles corresponding to the 110 A particles seen in intact chloroplasts. A model is presented suggesting that chloroplast lamellar membranes have a binary structure, which digitonin splits into two components. The two membrane fragments have different structures, revealed by freeze-etching, and different photochemical and biochemical functions.     

Evidence for two-step processing of nuclear-encoded chloroplast proteins during membrane assembly

A plastome (chloroplast genome) mutant of tobacco, lutescens-1, displays abnormal degradation of the chloroplast-encoded polypeptides which form the core complex of photosystem II (PSII). Two nuclear-encoded proteins (present in polymorphic forms), which normally function in the water oxidation process of PSII, accumulate as larger size-class polypeptides in mutant thylakoid membranes. These accumulated proteins are intermediate in size between the full-length primary protein synthesized in the cytoplasm and the proteolytically processed mature polypeptides. Trypsin treatment of unstacked mutant thylakoids and of inside-out vesicle (PSII-enriched) preparations indicated that the intermediate size forms were correctly localized on the inner surface of the thylakoid membrane, but not surface-exposed in the same way as the mature proteins. Only one of the intermediate size-class proteins could be extracted by salt washes. We interpret these data to be consistent with the idea that the two imported proteins that function in the water oxidation step of photosynthesis and are localized in the loculus (the space within the thylakoid vesicles) undergo two-step processing. The second step in proteolytic processing may be related to transport through a second membrane (the first transport step through the chloroplast envelope having been completed); this step may be arrested in the mutant due to the absence of the PSII core complex.     

Conformation and orientation of chlorophyll-proteins in photosystem I by circular dichroism and polarized infrared spectroscopies

The protein conformation and orientation of Photosystem I (PS I) particles have been investigated by a combination of ultraviolet circular dichroism and polarized infrared spectroscopies. These PS I particles have been studied before and after reconstitution in phosphatidylcholine vesicles. The native state of the pigments of PS I was characterized by monitoring the low-temperature fluorescence emission spectra as well as the visible CD and linear dichroism spectra at room temperature. Computed analysis of the ultraviolet CD spectra of PS I complex indicates that the secondary structure of the protein is largely α-helical (52 ± 4%) with a very low amount of β-structure. Polarized infrared difference spectra of oriented PS I show a significant orientation of these α-helical segments with the α-helix axes tilted on the average at approx. 35° from the membrane normal.     

Molecular evolution of the MAGUK family in metazoan genomes

Background  

Development, differentiation and physiology of metazoans all depend on cell to cell communication and subsequent intracellular signal transduction. Often, these processes are orchestrated via sites of specialized cell-cell contact and involve receptors, adhesion molecules and scaffolding proteins. Several of these scaffolding proteins important for synaptic and cellular junctions belong to the large family of membrane-associated guanylate kinases (MAGUK). In order to elucidate the origin and the evolutionary history of the MAGUKs we investigated full-length cDNA, EST and genomic sequences of species in major phyla.     

Extracellular identification of a processed type II ComR/ComS pheromone of Streptococcus mutans

The competence-stimulating peptide (CSP) and the sigX-inducing peptide (XIP) are known to induce Streptococcus mutans competence for genetic transformation. For both pheromones, direct identification of the native peptides has not been accomplished. The fact that extracellular XIP activity was recently observed in a chemically defined medium devoid of peptides, as mentioned in an accompanying paper (K. Desai, L. Mashburn-Warren, M. J. Federle, and D. A. Morrison, J. Bacteriol. 194:3774-3780, 2012), provided ideal conditions for native XIP identification. To search for the XIP identity, culture supernatants were filtered to select for peptides of less than 3 kDa, followed by C(18) extraction. One peptide, not detected in the supernatant of a comS deletion mutant, was identified by tandem mass spectrometry (MS/MS) fragmentation as identical to the ComS C-terminal sequence GLDWWSL. ComS processing did not require Eep, a peptidase involved in processing or import of bacterial small hydrophobic peptides, since eep deletion had no inhibitory effect on XIP production or on synthetic XIP response. We investigated whether extracellular CSP was also produced. A reporter assay for CSP activity detection, as well as MS analysis of supernatants, revealed that CSP was not present at detectable levels. In addition, a mutant with deletion of the CSP-encoding gene comC produced endogenous XIP levels similar to those of a nondeletion mutant. The results indicate that XIP pheromone production is a natural phenomenon that may occur in the absence of natural CSP pheromone activity and that the heptapeptide GLDWWSL is an extracellular processed form of ComS, possibly the active XIP pheromone. This is the first report of direct identification of a ComR/ComS pheromone.     

A combination of techniques proves useful in the development of nuclear markers in the newt genus Triturus

To increase the number of markers available for study of phylogeny and phylogeography in the newt genus Triturus, we developed and tested 59 primer pairs using three different techniques. Primers were obtained from published sources, by designing exon-primed intron-crossing primers and from randomly cloned anonymous nuclear DNA fragments. Successful polymerase chain reaction products were cloned and sequenced. Five fragments were successfully amplified and sequenced for six species of Triturus: intron 7 of the β-fibrinogen gene (βfibint7), third intron of the calreticulin gene (CalintC), the 11th intron of the α-subunit of the platelet derived growth factor receptor (PDGFRα) and two anonymous markers (Cri1 and Cri4). The average percentage species divergence across all the markers is low (c. 3%), compared to what has been found in mitochondrial DNA (25-30%).     

Inhibition of photophosphorylation by tentoxin, a cyclic tetrapeptide

Tentoxin, a fungal toxin which is known to cause seedling chlorosis, was found to inhibit cyclic photophosphorylation but not reversible proton accumulation by isolated chloroplasts. The toxin, at low concentrations, also inhibited coupled electron flow (in the presence of ADP and phosphate) but did not effect basal electron flow (in the absence of ADP and phosphate) or uncoupled electron transport. It is suggested that tentoxin is an energy transfer inhibitor which acts at the terminal steps of ATP synthesis.