Biology subjects

Niemeyer, J.

Publications and source records attributed to Niemeyer, J..

5 recordsLinked to original sources

bioRxiv2025

The proxiomes of CDJ5 and PGRL1 in Chlamydomonas reinhardtii overlap depending on the ability of CDJ5 to bind a 4Fe-4S cluster

The chloroplast chaperone HSP70B from Chlamydomonas reinhardtii works with the J-domain co-chaperones CDJ1 to CDJ6. CDJ1 delivers unfolded proteins, while CDJ2 delivers VIPP1 as substrate to HSP70B. CDJ3 to CDJ5 contain 4Fe-4S clusters in addition to the J domain, but their function is unknown. To investigate the function of CDJ5, we performed TurboID - mediated proximity labeling on wild-type CDJ5 (CDJ5-WT) and CDJ5 mutants with an impaired ability to stimulate HSP70Bs ATPase activity (CDJ5-AAA) or bind a 4Fe-4S cluster (CDJ5- SSS). Our results revealed that the proxiomes of all CDJ5 variants contained HSP70B and HSP90C. Furthermore, the proxiomes of CDJ5-WT and CDJ5-AAA overlapped extensively but differed from that of CDJ5-SSS, suggesting that the localization of CDJ5 to a chloroplast microcompartment depends on the presence of a functional 4Fe-4S cluster or its redox state. The CDJ5-WT and CDJ5-AAA proxiomes were enriched with proteins that regulate photosynthetic electron flow or are involved in the biogenesis of thylakoid membrane protein complexes and pigments. These proteins were also present in the proxiome of PGRL1 found in the CDJ5-WT and CDJ5-AAA proxiomes. Overall, our results suggest that CDJ5 acts with HSP70B/HSP90C via its 4Fe-4S cluster to regulate photosynthetic electron flow and thylakoid membrane protein complex biogenesis. HighlightDepending on its 4Fe-4S cluster, HSP70B co-chaperone CDJ5 localizes to a chloroplast microcompartment defined by PGRL1 where it could act in regulating photosynthetic electron flow and thylakoid membrane protein complex biogenesis.

plant biology↗

bioRxiv2022

CLPB3 is required for the removal of chloroplast protein aggregates and for thermotolerance in Chlamydomonas

In the cytosol of plant cells, heat-induced protein aggregates are resolved by ClpB/Hsp100 family member HSP101, which is essential for thermotolerance. For chloroplast family member CLPB3 this is less clear with controversial reports on its role in conferring thermotolerance. To shed light onto this issue, we have characterized two Chlamydomonas reinhardtii clpb3 mutants. We show that chloroplast CLPB3 is required for resolving heat-induced protein aggregates containing stromal TIG1 and the small heat shock proteins HSP22E/F in vivo and for conferring thermotolerance under heat stress. Although CLPB3 accumulates to similarly high levels as stromal HSP70B under ambient conditions, we observed no prominent constitutive phenotypes. However, we found decreased accumulation of the ribosomal subunit PRPL1 and increased accumulation of the stromal protease DEG1C in the clpb3 mutants, suggesting that reduction in chloroplast protein synthesis capacity and increase in protease capacity may compensate for loss of CLPB3 function. Under ambient conditions, CLPB3 was distributed throughout the chloroplast but reorganized into stromal foci upon heat stress, which mostly disappeared during recovery. CLPB3 foci were localized next to signals from HSP22E/F, originating largely to the thylakoid membrane occupied area. This suggests a possible role for CLPB3 in disentangling protein aggregates from the thylakoid membrane system. HighlightChloroplast CLPB3 in Chlamydomonas is required for resolving heat-induced protein aggregates and this activity confers thermotolerance under severe heat stress. During heat stress, CLPB3 organizes into stromal foci located next to the thylakoid membrane system, indicating a role for CLPB3 in disentangling protein aggregates from there.

plant biology↗

bioRxiv2021

Production and secretion of functional full-length SARS-CoV-2 spike protein in Chlamydomonas reinhardtii

The spike protein is the major protein on the surface of coronaviruses. It is therefore the prominent target of neutralizing antibodies and consequently the antigen of all currently admitted vaccines against SARS-CoV-2. Since it is a 1273-amino acids glycoprotein with 22 N-linked glycans, the production of functional, full-length spike protein was limited to mammalian and insect cells, requiring complex culture media. Here we report the production of full-length SARS-CoV-2 spike protein - lacking the C-terminal membrane anchor - as a secreted protein in the prefusion-stabilized conformation in the unicellular green alga Chlamydomonas reinhardtii. We show that the spike protein is efficiently cleaved at the furin cleavage site during synthesis in the alga and that cleavage is abolished upon mutation of the multi-basic cleavage site. We could enrich the spike protein from culture medium by ammonium sulfate precipitation and demonstrate its functionality based on its interaction with recombinant ACE2 and ACE2 expressed on human 293T cells. Chlamydomonas reinhardtii is a GRAS organism that can be cultivated at low cost in simple media at a large scale, making it an attractive production platform for recombinant spike protein and other biopharmaceuticals in low-income countries.

synthetic biology↗

bioRxiv2020

Real-time monitoring of subcellular H2O2 distribution in Chlamydomonas reinhardtii

H2O2 has been recognized as an important signaling molecule in plants. We sought to establish a genetically encoded, fluorescent H2O2 sensor that allows H2O2 monitoring in all major subcompartments of a Chlamydomonas cell. To this end we engineered the hypersensitive H2O2 sensor, roGFP2-Tsa2{Delta}CR, as a genetic part for the Chlamydomonas Modular Cloning toolbox. Using previously generated parts, together with new ones, we constructed modules and devices that target the sensor to the cytosol, nucleus, mitochondrial matrix, chloroplast stroma, thylakoid lumen, and ER. The sensor was functional in all compartments, except for the ER where it was fully oxidized. Employing our novel sensors, we show that H2O2 produced by photosynthetic linear electron transport (PET) in the stroma leaks into the cytosol but only reaches other subcellular compartments if produced under non-physiological conditions. Our results thus imply the establishment of steep intracellular H2O2 gradients under normal physiological conditions and suggest that the cytosolic complement of H2O2 scavenging enzymes effectively limits H2O2 diffusion. Furthermore, in heat stressed cells, we show that cytosolic H2O2 levels closely mirror temperature up- and downshifts and are independent from PET. We anticipate that these sensors will greatly facilitate future investigations into H2O2 biology in algal and plant cells.

plant biology↗

bioRxiv2020

Structural basis for VIPP1 oligomerization and maintenance of thylakoid membrane integrity

Vesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis and maintenance of thylakoid membranes, which transform light into life. However, it is unknown how VIPP1 performs its vital membrane-shaping function. Here, we use cryo-electron microscopy to determine structures of cyanobacterial VIPP1 rings, revealing how VIPP1 monomers flex and interweave to form basket-like assemblies of different symmetries. Three VIPP1 monomers together coordinate a non-canonical nucleotide binding pocket that is required for VIPP1 oligomerization. Inside the rings lumen, amphipathic helices from each monomer align to form large hydrophobic columns, enabling VIPP1 to bind and curve membranes. In vivo point mutations in these hydrophobic surfaces cause extreme thylakoid swelling under high light, indicating an essential role of VIPP1 lipid binding in resisting stress-induced damage. Our study provides a structural basis for understanding how the oligomerization of VIPP1 drives the biogenesis of thylakoid membranes and protects these life-giving membranes from environmental stress.

plant biology↗