Hirokazu Katoh

Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1

NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated NDH-1MS, was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

Rice SNF2 family helicase ENL1 is essential for syncytial endosperm development.

The endosperm of cereal grains represents the most important source of human nutrition. In addition, the endosperm provides many investigatory opportunities for biologists because of the unique processes that occur during its ontogeny, including syncytial development at early stages. Rice endospermless 1 (enl1) develops seeds lacking an endosperm but carrying a functional embryo. The enl1 endosperm produces strikingly enlarged amoeboid nuclei. These abnormal nuclei result from a malfunction in mitotic chromosomal segregation during syncytial endosperm development. The molecular identification of the causal gene revealed that ENL1 encodes an SNF2 helicase family protein that is orthologous to human Plk1-Interacting Checkpoint Helicase (PICH), which has been implicated in the resolution of persistent DNA catenation during anaphase. ENL1-Venus (enhanced yellow fluorescent protein (YFP)) localizes to the cytoplasm during interphase but moves to the chromosome arms during mitosis. ENL1-Venus is also detected on a thread-like structure that connects separating sister chromosomes. These observations indicate the functional conservation between PICH and ENL1 and confirm the proposed role of PICH. Although ENL1 dysfunction also affects karyokinesis in the root meristem, enl1 plants can grow in a field and set seeds, indicating that its indispensability is tissue-dependent. Notably, despite the wide conservation of ENL1/PICH among eukaryotes, the loss of function of the ENL1 ortholog in Arabidopsis (CHR24) has only marginal effects on endosperm nuclei and results in normal plant development. Our results suggest that ENL1 is endowed with an indispensable role to secure the extremely rapid nuclear cycle during syncytial endosperm development in rice.

Cloning of the cotA gene of Synechococcus PCC7942 and complementation of a cotA-less mutant of Synechocystis PCC6803 with chimeric genes of the two strains

AbstractcotA, a homologue of cemA that encodes a chloroplast envelope membrane protein, was cloned from Synechococcus PCC7942. The gene encodes a protein of 421 amino acids, which is similar in size to CotA of Synechocystis PCC6803 and CemA of liverwort and Chlamydomonas. There was significant sequence homology among these CotA and CemA in the C-terminal region but the homology was low in the N-terminal region. Sequencing of Synechococcus DNA in the cotA region revealed two other genes downstream of cotA, one of which is homologous to cobP and could be cotranscribed with cotA. A mutant (M48) was constructed by inactivating cotA in the wild-type (WT) Synechococcus. The mutant showed the same characteristics as the cotA-deletion mutant of Synechocystis (M29) and was unable to grow in a low sodium medium or at acidic pH under aeration with 3% CO2in air (v/v). Synechococcus cotA did not comple-ment M29. Three chimeric cotA genes of the two cyanobacterial strains were constructed. One of these chimeric genes strongly and the other two weakly complemented the mutant.

Structure and Function of Cema Homologue (PXCA) in Cyanobacteria

The cemA (ycf10) gene codes for a chloroplast envelope membrane protein [1] and is conserved in higher and lower plants and in algae [2–8]. CemA in higher plants consists of 229 to 231 amino-acids [2–4] whereas that in liverwort (Marchantia) [5] and Chlamydomonas [6] is much larger and consists of 434 and 500 amino-acids, respectively. Recent sequencing of whole chloroplast genomes of Porphyra [7] and Chlorella [8] revealed that cemA in these algae encodes proteins of 278 and 264 amino-acids, respectively. The function of CemA is not known. Rolland et al [6] have constructed mutants by disrupting cemA in Chlamydomonas. They showed that the disruption of the gene led to increased light sensitivity and affected CO2-dependent photosynthesis and inorganic carbon uptake.

Cema Homologue in Cyanobacteria (PXCA) Involved in Proton Exchange

When suspension of cyanobacterial cells are illuminated, there is an acidification of the medium followed by an alkalization [1-4]. Both acidification and alkalization are specifically stimulated by Na+ The acidification was assumed to be due to a light-dependent uptake of CO2 that is converted to (HCO_3^ -) [2,3] and the alkalization due to extrusion of OH- produced as a result of conversion of (HCO_3^ -) to CO2 that is fixed by photosynthesis [1]. Ambiguity, however, remains on the source of protons or hydroxyl ions extruded in the light and electron transport involved in the proton extrusion is not yet known.