Abstract Isoamylases are debranching enzymes that hydrolyze α-1,6 linkages in α-1,4/α-1,6–linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although precise manner which influence starch is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) identified from potato. The expression patterns genes consistent with possibility all play roles synthesis. Analysis predicted sequences...

The Bundle sheath defective2 (Bsd2) gene is required for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulation in maize. Using a Mutator transposable element as molecular probe, we identified tightly linked restriction fragment length polymorphism that cosegregated with the bsd2-conferred phenotype. This was cloned, and sequences flanking insertion were used to screen maize leaf cDNA library. full-length clone isolated this screen, show an abundant 0.6-kb transcript could be...

The recently discovered potato tuber ( Solanum tuberosum ) α-glucan, water dikinase (GWD) (formerly known as R1) catalyzes the phosphorylation of starch by a dikinase-type reaction mechanism in which β-phosphate ATP is transferred to either C-6 or C-3 position glucosyl residue starch. In present study, we found that GWD enzyme inactive oxidized form, accompanied formation specific intramolecular disulfide bond determined disulfide-linked peptide mapping. regulatory properties this linkage...

Cross-presentation is the presentation by MHC class I of antigenic peptides from exogenous proteins that have been internalized and processed professional antigen-presenting cells, e.g. dendritic cells. We investigated influence particle size antigen load on cross-presentation following delivery microspheres (MS). small particles (0·8-μm) sensitive to proteasome inhibition blockade endoplasmic reticulum-resident complex export, whereas larger (aggregated clumps 0·8-μm MS) resistant these...

The homologous proteins Oxa1, YidC, and Alb3 mediate the insertion of membrane in mitochondria, bacteria, chloroplast thylakoids, respectively. Depletion YidC Escherichia coli affects integration every protein studied, has been shown previously to be required for a signal recognition particle (SRP)-dependent protein, Lhcb1, thylakoids. In this study we have analyzed "global" role thylakoid proteins. We show that two chlorophyll-binding proteins, Lhcb4.1 Lhcb5, is almost totally blocked by...

Two distinct mechanisms have been previously identified for the transport of proteins across chloroplast thylakoid membrane, one which is unusual in that neither soluble factors nor ATP are required; system requires only transthylakoidal ΔpH. We examined this mechanism by studying properties its substrates: extrinsic 23-kDa protein (23K) photosystem II. Previous work has shown can be transported into isolated thylakoids as full-length precursor protein; we show stromal import intermediate...

Translocation across the thylakoid membrane of recently identified photosystem I polypeptide, PSI-N, has been analyzed in pea (Pisum sativum) and barley (Hordeum vulgare). PSI-N from is synthesized cytosol with a bipartite presequence similar structural terms to those other cytosolically proteins routed lumen. In vitro reconstitution assays demonstrate that translocation into thylakoids absolutely dependent on transthylakoidal delta pH, but nucleotide triphosphates are not required;...

The signal recognition particle (SRP) is a ubiquitous system for the targeting of membrane and secreted proteins. chloroplast SRP (cpSRP) unique among SRPs in that it possesses no RNA functional post-translational as well co-translational targeting. We have expressed purified two components Arabidopsis thaliana involved transport: cpSRP54 chloroplast-specific protein, cpSRP43. Recombinant cpSRP supports efficient vitro insertion pea preLhcb1 into isolated thylakoid membranes. stable...

The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa protein novel repeats zinc fingers described as interaction domains. interacts specifically in yeast vitro with NCED4, putative polyene chain carotenoid dioxygenase, both NCED4 accumulate chloroplast stroma. Metabolic...

The photosystem I subunit PsaK spans the thylakoid membrane twice, with N and C termini both located in lumen. insertion mechanism of a protein adopting this type topology has not been studied before, we have used vitro assays to determine requirements for into thylakoids. inserts high efficiency show that one transmembrane span (the C-terminal region) can insert independently other, indicating "hairpin"-type is essential. Insertion does require stromal extract, signal recognition particle...

A novel polypeptide with an apparent molecular mass of 9 kDa was detected after sodium dodecyl sulphate-polyacrylamide gel electrophoresis Arabidopsis photosystem I (PSI) and N-terminally sequenced. Corresponding cDNA clones encode a precursor protein 140 amino acid residues which imported into isolated intact chloroplasts processed to the mature protein, designated PSI-O. The has two transmembrane helices calculated 10104 Da. PSI-O also shown be present in PSI from barley spinach,...

We developed a rapid and reliable technique for specifically staining intact chloroplasts using the fluorescent dye carboxyfluorescein diacetate. Intact, import-competent were isolated simply rapidly from soil-grown Arabidopsis thaliana plants, with yields of 20 ± 5 μg chlorophyll per g FW, greater than previously reported Arabidopsis. Traditional chloroplast isolation buffers sometimes contain low concentrations (<10 mM) sodium ascorbate as general-purpose anti-oxidant, but we found that...

An Arabidopsis cDNA coding for a previously uncharacterized isoform of inorganic pyrophosphatase was isolated. It used to complement an E. coli mutant, demonstrating that it coded active enzyme. MgCl 2 necessary the protein's activity, whilst NaF inhibited it. The K m pyrophosphate and pH optimum protein determined. gene this expressed in all tissues, its expression rosette leaves induced by incubation on metabolizable sugars. In vitro import experiments demonstrated could be imported into...

The biogenesis of several bacterial polytopic membrane proteins has been shown to require signal recognition particle (SRP) and protein transport machinery, one such protein, the major light-harvesting chlorophyll-binding (LHCP) exhibits these requirements in chloroplasts. In this report we have used vitro insertion assays analyze four additional members chlorophyll-a/b-binding family. We show that two members, Lhca1 Lhcb5, display an absolute requirement for stroma, nucleoside...

Two imported thylakoid membrane proteins, PSII‐X and PSII‐W, are synthesised with cleavable N‐terminal signal peptides that closely resemble those of Sec‐dependent lumenal proteins. In this report we have reconstituted the insertion pre‐PSII‐X pre‐PSII‐W into isolated thylakoids. We show does not require either nucleoside triphosphates or stromal extracts, both which required for Sec‐ recognition particle (SRP)‐dependent targeting mechanisms. Insertion is furthermore unaffected by protease...

The photosystem I (PSI) complex in higher plants contains eight nuclear-encoded subunits, of which two (PSI-F and -N) are synthesized with bipartite presequences containing cleavable thylakoid transfer sequences. Previous studies on four other chloroplast proteins bearing have shown that they transported across the membrane by distinct mechanisms. One mechanism is delta pH-dependent hence sensitive to uncouplers, whereas inhibited azide. We show PSI-F targeted latter pathway, since its...

Two-pore domain potassium (K2P) channels play fundamental roles in cellular processes by enabling a constitutive leak of from cells which they are expressed, thus influencing membrane potential and activity. Hence, regulation these is critical importance to function. A key regulatory mechanism K2P the control their cell surface expression. Membrane protein delivery retrieval controlled passage through secretory endocytic pathways, post-translational modifications regulate progression...

The nuclear psbY gene (formerlyycf32) encodes two distinct single-spanning chloroplast thylakoid membrane proteins in Arabidopsis thaliana. After import into the chloroplast, precursor protein is processed to a polyprotein which each “mature” preceded by an additional hydrophobic region; we show that these regions function as signal peptides are cleaved after insertion membrane. Inhibition of first or second cleavage reaction enlargement −1 residues leads case accumulation...

We have cloned an Arabidopsis cDNA encoding a novel thylakoid lumen protein, P17.4, that has been previously isolated from extracts of spinach chloroplasts. The protein is synthesised with bipartite presequence containing Sec‐type lumen‐targeting signal peptide and the precursor imported into pea encoded homologous to Anabaena essential for correct glycolipid localisation, also related at least 16 unassigned open reading frames in Synechocystis . This family proteins characterised by...