Biology subjects

Fukuzawa, H.

Publications and source records attributed to Fukuzawa, H..

6 recordsLinked to original sources

bioRxiv2025

A nuclear CobW/WW-domain factor represses the CO2-concentrating mechanism in the green alga Chlamydomonas reinhardtii

Microalgae induce a CO2-concentrating mechanism (CCM) to maintain photosynthesis when dissolved CO2 is limited, but how this energy-intensive system is suppressed when CO2 levels rise has remained unclear. The CCM consumes 15-30% of photosynthetically-generated ATP, making its regulation critical for cellular energy balance. Here, we identify a nuclear repressor of the CCM in the green alga Chlamydomonas reinhardtii. A pull-down screen for interacting partners of the master activator CCM1/CIA5 revealed an uncharacterized protein that co-purifies with CCM1 even after high-salt washes. This protein, designated CCM1-binding protein 1 (CBP1), combines a CobW/CobW_C GTP-binding metallochaperone module with a WW domain characteristic of protein-protein interactions. CBP1 colocalizes with CCM1 in the nucleus regardless of CO2 conditions, and the two proteins interact in vivo. CRISPR/Cas9-based disruption of CBP1 does not affect growth or CCM induction under CO2 limitation but derepresses 27 of 41 CCM1-dependent low-CO2 inducible genes under high-CO2 conditions. These include the periplasmic and intracellular carbonic anhydrases (CAH1, LCIB) and inorganic carbon transporters/channels (LCIA, LCI1, BST1, BST3). Consistently, cbp1 mutants accumulate higher levels of CAH1 and LCIB proteins and exhibit a 40% increase in inorganic carbon affinity under high-CO2 conditions; this phenotype is rescued by CBP1 complementation or by acetazolamide treatment. These results demonstrate that CBP1 prevents unnecessary CCM activity when CO2 is abundant, acting upstream of both transporter/channel and carbonic anhydrase modules. Our findings suggest a regulatory mechanism potentially linking zinc-dependent protein chemistry to CCM gene repression, providing insights into energy-efficient CO2 sensing in aquatic photosynthetic organisms. Significance statementAlgae flourish by activating a CO2-concentrating mechanism (CCM) when dissolved CO2 is limited but must deactivate it when CO2 levels increase to conserve energy. We have identified the nuclear protein that functions as this long sought "off switch" in the model green alga. Deletion of this protein causes cells to overproduce CCM transporters and enzymes, maintaining CCM activity even under high CO2 conditions, demonstrating its essential role in suppressing the system when carbon is abundant. This finding illuminates how algae balance energy consumption with carbon capture and offers a new target for engineering strains that fix CO2 more efficiently for biofuel production or climate-mitigation technologies.

plant biology↗

bioRxiv2024

Periplasmic carbonic anhydrase CAH1 contributes to high inorganic carbon affinity in Chlamydomonas reinhardtii

Carbonic anhydrase (CA), an enzyme conserved across species, is pivotal in the interconversion of inorganic carbon (Ci; CO2 and HCO3-). Compared to the well-studied intracellular CA, the specific role of extracellular CA in photosynthetic organisms is still not well understood. In the green alga Chlamydomonas reinhardtii, CAH1, located at the periplasmic space, is strongly induced under CO2-limiting conditions by the Myb transcription factor LCR1. While it has been observed that the lcr1 mutant shows decreased Ci-affinity, the detailed mechanisms behind this phenomenon are yet to be elucidated. In this study, we aimed to unravel the LCR1-dependent genes essential for maintaining high Ci-affinity. To achieve this, we identified a total of 12 LCR1-dependent inducible genes under CO2-limiting conditions, focusing specifically on the most prominent ones - CAH1, LCI1, LCI6, and Cre10.g426800. We then created mutants of these genes using the CRISPR-Cas9 system, all from the same parental strain, and compared their Ci-affinity. Contrary to earlier findings (Van and Spalding, 1999) that reported no reduction in Ci-affinity in the cah1 mutant, our newly created cah1-1 mutant exhibited a significant decrease in Ci-affinity under high HCO3-/CO2-ratio conditions. Additionally, when we treated wild-type cells with a CA inhibitor with low membrane permeability, a similar reduction in Ci-affinity was observed. Moreover, the addition of exogenous CA to the cah1 mutant restored the decreased Ci-affinity. These results, highlighting the crucial function of the periplasmic CAH1 in maintaining high Ci-affinity in Chlamydomonas cells, provide new insights into the functions of periplasmic CA in algal carbon assimilation. One-sentence summaryCAH1, a periplasmic carbonic anhydrase in Chlamydomonas reinhardtii, plays a crucial role in maintaining a high affinity for inorganic carbon, particularly under CO2-limiting conditions.

plant biology↗

bioRxiv2022

Proton-pumping rhodopsins in marine diatoms

Diatoms are a major phytoplankton group responsible for about 20% of Earths primary production. They carry out photosynthesis inside the plastid, an organelle obtained through eukaryote-eukaryote endosymbiosis. Recently, microbial rhodopsin, a photoreceptor distinct from chlorophyll-based photosystems, has been identified in certain diatoms. However, the physiological function of diatom rhodopsin is not well understood. Here we show that the diatom rhodopsin acts as a light-driven proton pump and localizes to the outermost membrane of the four membrane-bound complex plastids. Heterologous expression techniques were used to investigate the protein function and subcellular localization of diatom rhodopsin. Using model simulations, we further evaluated the physiological role of the acidic pool in the plastid produced by proton-transporting rhodopsin. Our results propose that the rhodopsin-derived acidic pool may be involved in a photosynthetic CO2-concentrating mechanism and assist CO2 fixation in diatom cells.

microbiology↗

bioRxiv2021

Excretion of triacylglycerol as a matrix lipid facilitating apoplastic accumulation of a lipophilic metabolite shikonin.

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. The mechanism of secretion remains largely unknown, because hydrophobic metabolites, which may form oil droplets or crystals in cytosol, inducing cell death, cannot be directly secreted by transporters. Moreover, some secondary metabolic lipids react with cytosolic components leading to their decomposition. Lipophilic metabolites should thus be solubilized by matrix lipids and compartmentalized by membrane lipids. The mechanism of lipophilic metabolite secretion was assessed using shikonin, a red naphthoquinone lipid, in Lithospermum erythrorhizon. Cell secretion of shikonin also involved the secretion of about 30% of triacylglycerol (TAG), composed predominantly of saturated fatty acids. Shikonin production was associated with the induction of large amounts of the membrane lipid phosphatidylcholine. Together with in vitro reconstitution, these findings suggest a novel role for TAG as a matrix lipid for the secretion of lipophilic metabolites.

plant biology↗

bioRxiv2020

Chlamydomonas reinhardtii Tubulin-Gene Disruptants for Efficient Isolation of Strains Bearing Novel Tubulin Mutations

The single-cell green alga Chlamydomonas reinhardtii possesses two -tubulin genes (tua1 and tua2) and two {beta}-tubulin genes (tub1 and tub2), with the two genes in each pair encoding identical amino acid sequences. Here, we used an aphVIII gene cassette insertional library to establish eight disruptants with defective tua2, tub1, or tub2 expression. None of the disruptants exhibited apparent defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants of C. reinhardtii have been reported to date, we then used our disruptants, together with a tua1 disruptant obtained from the Chlamydomonas Library Project (CLiP), to isolate novel tubulin-mutants resistant to the anti-tubulin agents propyzamide and oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different -tubulin mutations and 24 strains bearing 12 different {beta}-tubulin mutations. Some of these mutations are known to confer drug resistance in human cancer cells. Thus, single-tubulin-gene disruptants are an efficient means of isolating novel C. reinhardtii tubulin mutants. IMPORTANCEChlamydomonas reinhardtii is a useful organism for the study of tubulin function; however, only five kinds of tubulin mutations have been reported to date. This scarcity is partly due to C. reinhardtii possessing two tubulin genes each for - and {beta}-tubulin. Here, we obtained several strains in which one of the - or {beta}-tubulin genes was disrupted, and then used those disruptants to isolate 32 strains bearing 19 mostly novel tubulin mutations that conferred differing degrees of resistance to two anti-tubulin compounds. The majority of the tubulin mutations were located outside of the drug-binding sites in the three-dimensional tubulin structure, suggesting that structural changes underlie the drug resistance conferred by these mutations. Thus, single-tubulin-gene disruptants are an efficient means of generating tubulin mutants for the study of the structure-function relationship of tubulin and for the development of novel therapies based on anti-tubulin agents.

cell biology↗

bioRxiv2018

Inner lumen proteins stabilize doublet microtubules in cilia/flagella

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although it is known that microtubules in cilia are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dynein and intraflagellar transport. To address this question, we identified a new class of microtubule-associated proteins, named FAP45 and FAP52, in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we established that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These inner protrusions are located near the inner junctions of doublet microtubules and lack of FAP45, FAP52, and FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrated that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

cell biology↗