Aurea C. Vasconcelos

Physical association of KAB1 with plant K+ channel alpha subunits.

K+ channel proteins contain four alpha subunits that align along a central axis perpendicular to membranes and form an ion-conducting pore. Recent work with K+ channels native to animal membranes has shown that at least some members of this protein family also have four beta subunits. These structural components of the holoenzyme each form tight associations with the cytoplasmic portion of an alpha subunit. We have cloned an Arabidopsis cDNA (KAB1) that encodes a polypeptide sharing 49% amino acid identity with animal K+ channel beta subunits. In this study, we provide experimental evidence that the KAB1 polypeptide forms a tight physical association with the Arabidopsis K+ channel alpha subunit, KAT1. An affinity-purified KAB1 fusion protein was immobilized to a support resin and shown to sequester selectively the KAT1 polypeptide. In addition, polyclonal antibodies raised against KAB1 were shown to immunoprecipitate the KAT1 polypeptide as a KAT1-KAB1 protein complex. Immunoblot analysis demonstrated that KAB1 is expressed in Arabidopsis seedings and is present in both membrane and soluble protein fractions. The presence of KAB1 (a soluble polypeptide) in both soluble and membrane protein fractions suggests that a portion of the total amount of native KAB1 is associated with an integral membrane protein, such as KAT1. The presence of KAB1 in crude protein fractions prepared from different Arabidopsis plant organs was evaluated. High levels of KAB1 protein were present in flowers, roots, and leaves. Immunoblot analysis of protein extracts prepared from broad bean leaves indicated that the KAB1 expression level was 80-fold greater in guard cells than in mesophyll cells. Previous studies of the in situ transcription pattern of KAT1 in Arabidopsis indicated that this alpha subunit is abundantly present in leaves and, within the leaf, exclusively present in guard cells. Thus, KAB1 was determined to be expressed in plant organs (leaves) and cell types (guard cells) that are sites of KAT1 expression in the plant. The in situ expression pattern of KAB1 suggests that it may associate with more than one type of K+ channel alpha subunit. Sequence analysis indicates that KAB1 may function in plant K+ channels as an oxidoreductase. It is postulated that beta subunits native to animal K+ channels act as regulatory subunits through pyridine nucleotide-linked reduction of alpha polypeptides. Although the KAB1 primary structure is substantially different from that of animal beta subunits, amino acid motifs critical for this catalytic activity are retained in the plant beta subunit.

Evidence That Plant K+ Channel Proteins Have Two Different Types of Subunits

Plant K+ channel proteins have been previously characterized as tetramers of membrane-spanning [alpha] subunit polypeptides. Recent studies have identified a 39-kD, hydrophilic polypeptide that is a structural component of purified animal K+ channel proteins. We have cloned and sequenced an Arabidopsis thaliana cDNA encoding a 38.4-kD polypeptide that has a sequence homologous to the animal K+ channel [beta] subunit. Southern and northern analyses indicate the presence of a gene encoding this cDNA in the Arabidopsis genome and that its transcription product is present in Arabidopsis cells. To our knowledge, this is the first report to document the presence of K+ channel [beta] subunits in plants.

Isolation of a cDNA encoding mitochondrial citrate synthase from Arabidopsis thaliana

We isolated a cDNA clone from Arabidopsis thaliana encoding the TCA cycle enzyme, citrate synthase. The plant enzyme displays 48% and 44% amino acid residue similarity with the pig, and yeast polypeptides, respectively. Many proteins, including citrate synthase, which are destined to reside in organelles such as mitochondria and chloroplasts, are the products of the nucleocytoplasmic protein synthesizing machinery and are imported post-translationally to the site of function. We present preliminary investigations toward the establishment of an in vitro plant mitochondrial import system allowing for future studies to dissect this process in plants where the cell must differentiate between mitochondria and chloroplast and direct their polypeptides appropriately.

DNA duplication and chromosome structure in the Dinoflagellates

SummaryMembers of theDinopbyceae are characterized by having permanently condensed chromosomes throughout the cell cycle. At interphase the chromosomes appear to have bands perpendicular to the long axis of the chromosome with a periodicity of 127 nm. Each band is composed of 2.5 nm fibers and 9.0 nm granules coiled into a helix around a central core of 9.0 nm fibers. Chromosome uncoiling has been correlated with the uptake of3[H]-thymidine. As chromosomes enter the uncoiling phase of the cell cycle they appear less dense and reveal a number of fibrous extensions. At later stages chromosomes completely uncoil into elongate fibers 127 nm in width. Chromosome unwinding corresponds to the peak in the uptake of3[H]-thymidine. Chromosomes observed on either side of the peak possess the typical interphase banding. This study demonstrates, for the first time, the fine structural details of chromosome uncoiling during a specific phase of the cell cycle. A new model of the Dinoflagellate chromonema has been derived from this study.

Isolation and characterization of a cDNA encoding cytosolic fructose-1,6-bisphosphatase from spinach

Photosynthetic cells require fructose-1,6-bisphosphatase (FBPase) (EC 3.1.3.11) activity in the chloroplast as well as in the cytoplasm [ 1, 11 ]. In both cases the enzyme catalyzes the hydrolysis of fructose-l,6-bisphosphate (F1,6P2) to fructose6-phosphate (F6P) and inorganic phosphate (Pi). The chloroplast FBPase is an essential enzyme in the photosynthetic pathway of CO2 fixation into sugars. The cytosolic FBPase is required for the synthesis of sucrose from triosephosphate, the major form of reduced carbon exported from the chloroplast during photosynthesis. Due to the importance of the cytosolic FBPase in sucrose biosynthesis in photosynthetic tissues and in gluconeogenesis in non-photosynthetic tissues, a great effort has been focused on understanding the regulatory mechanism of the enzyme Ireviewed in 20, 21]. Nevertheless, most biochemical information, like reactive sites, came from animal systems [7-10, 14, 24]. In this paper, we report the first DNA sequence of a plant cytosolic FBPase. We have isolated and characterized a cDNA encoding a cytosolic FBPase from spinach, compared the amino acid sequence derived from the nucleotide sequence with other published FBPases, and analyzed the possible functional amino acid residues on the basis of structural studies [7-10]. These data have been presented previously in abstract form [6]. A gene encoding yeast FBPase was obtained from Dr D.G. Fraenkel [19]. A 600 bp Barn HI-Xba I fragment from the center of the 2.5 kb yeast genomic clone was used as a hybridization probe to screen 300 000 clones in a spinach leaf cDNA library constructed in the expression vector 2gtl l [22]. The cDNA inserts were subcloned into the Eco RI site of the plasmid vector pGEM3 (Promega, Madison, WI). To sequence both strands as well as to obtain overlapped fragments, a series of unidirectional deletions were generated [5] from two recombinant plasmids, pFBPY and pFPBI, carrying the cDNA insert in the forward and inverted orientation, respectively. The inserts were sequenced by the dideoxy chain termination method of Sanger etal. [18], using the Sequenase system (USB, Cleveland, OH) as described by the manufacturer. Figure 1 shows the complete nucleotide sequence of 1585 bp of a cDNA clone encoding a cytosolic FBPase and the deduced amino acid

Euploidy in Ricinus. I. Euploidy and gene dosage effects on cellular proteins

Investigation of an euploid series of castor bean, Ricinus communis L., consisting of haploid, diploid, and tetraploid individuals, was performed to determine the value of such a series in studying the biochemical consequences of genome multiplication. The effects of euploidization of the nuclear genome on the biosynthesis of cellular proteins were examined. Extracts of total soluble proteins from 10-day-old leaves of all three ploidy levels examined by electrophoresis on polyacrylamide gels revealed no difference in the complement of proteins present; however, differences in intensity of several protein bands were detected. Analysis of esterase isozyme activity by isoelectric focusing revealed both increases and decreases in the activity levels of individual isozyme variants in response to changes in ploidy levels. Results from this analysis are discussed in terms of possible regulatory mechanisms active in the regulation of duplicated genes.

THE INTRAMEMBRANOUS PARTICLE PROFILE OF THE PARAMYLON MEMBRANE DURING PARAMYLON SYNTHESIS IN EUGLENA (EUGLENOPHYCEAE)

Paramylon is the β‐1, 3‐glucan storage product in euglenoid algae. It is a fibrous crystal that occurs as membrane‐bound granules in the cytosol. The role of the surrounding membrane in paramylon synthesis was investigated by the use of freeze‐etch electron microscopy. When Euglena gracilis Klebs strain Z (Pringsheim) cells were frozen in supercooled liquid nitrogen, the fracture plane primarily was throuh the paramylon membrane. A large intramembranous particle (IMP, mean diam range 5.6‐6.5 nm) and a small IMP (mean diam range 9.6‐10.3 nm) were predominant in both PF (protoplasmic fracture) and EF (exoplasmic fracture) faces of the paramylon membrane. During paramylon synthesis induction, the ratio of small to large IMPs increased in both fracture faces. The IMP density decreased in both fracture faces concomitant to paramylon synthesis increase. These changes in IMP profile and density suggest that the paramylon membrane is involved in the synthesis of paramylon.

Isolation of dictyosomes fromEuglena gracilis

SummaryA method for the isolation of dictyosomes fromEuglena gracilis Klebs strain Z (Pringsheim) is described. An extensive Golgi system, with the individual dictyosomes commonly containing ten to twenty cisternae is present. Log phase cells are broken in a French pressure cell at 105 to 120 kg/cm2 in a breaking mix containing sucrose, sorbitol and ficoll. Addition of 0.3% of glutaraldehyde or formaldehyde to the breaking mix increases the number of stacked cisternae present in the final preparation. In addition to membrane stacks, the fractions contain numerous smooth vesicles. Swollen cisternae, which are also present, may account for these vesicles. Three dictyosome-enriched fractions are obtained by centrifugation in a discontinuous sucrose gradient. Fractions differ morphologically in the degree of stacking of cisternae. Further identification of the membrane fractions was accomplished by measuring IDPase activities in each of the fractions. Inosine diphosphatase activity is enriched 8–10-fold relative to the initial homogenate. The highest IDPase activity was present in the fraction containing the greatest number of stacked cisternae.

Isolation of intact spinach chloroplasts in the CF-6 continuous-flow zonal rotor; Implications for membrane-bound organelles☆

Abstract Intact chloroplasts were separated from large volumes of spinach brei by continuous-flow zonal centrifugation in the CF-6 rotor in gradients of colloidal silica. Tests with the Z-15/Mark 10 rotor showed that the external membranes of chloroplasts were sensitive to high rotor speeds and especially to the bore of the fluid seal. Enlargement of the CF-6 seal channel diameter to 4 mm resulted in almost complete recovery of the intact chloroplasts.