Biology subjects

Patena, W.

Publications and source records attributed to Patena, W..

7 recordsLinked to original sources

bioRxiv2024

The Chlamydomonas reinhardtii CLiP2 mutant collection expands genome coverage with high-confidence disrupting alleles

Chlamydomonas reinhardtii (Chlamydomonas hereafter) is a powerful model organism for studies of photosynthesis, ciliary motility, and other cellular processes [1-4]. The CLiP library of mapped nuclear random insertion mutants [5,6] has accelerated progress for hundreds of laboratories in these fields by providing mutants in genes of interest. However, its value was limited by its modest coverage of the genome with high-confidence disruption alleles (46% of nuclear protein-coding genes with 1+ high-confidence allele in exons/introns; 12% of genes with 3+ alleles in exons/introns). Here we introduce the CLiP2 (Chlamydomonas Library Project 2) library, which greatly expands the number of available mapped high-confidence insertional mutants. The CLiP2 library includes 71,700 strains, covering 79% of nuclear protein-coding genes with 1+ high-confidence allele in exons/introns and 49% of genes with 3+ alleles in exons/introns. The mutants are available to the community via the Chlamydomonas Resource Center.

plant biology↗

bioRxiv2022

Systematic identification and characterization of novel genes in the regulation and biogenesis of photosynthetic machinery

Photosynthesis is central to food production and the Earths biogeochemistry, yet the molecular basis for its regulation remains poorly understood. Here, using high-throughput genetics in the model eukaryotic alga Chlamydomonas reinhardtii, we identify with high confidence (FDR<0.11) 70 previously-uncharacterized genes required for photosynthesis. We then provide a resource of mutant proteomes that enables functional characterization of these novel genes by revealing their relationship to known genes. The data allow assignment of 34 novel genes to the biogenesis or regulation of one or more specific photosynthetic complexes. Additional analysis uncovers at least seven novel critical regulatory proteins, including five Photosystem I mRNA maturation factors and two master regulators: MTF1, which impacts chloroplast gene expression directly; and PMR1, which impacts expression via nuclear-expressed factors. Our work provides a rich resource identifying novel regulatory and functional genes and placing them into pathways, thereby opening the door to a system-level understanding of photosynthesis. HighlightsO_LIHigh-confidence identification of 70 previously-uncharacterized genes required for photosynthesis C_LIO_LIProteomic analysis of mutants allows assignment of function to novel genes C_LIO_LICharacterization of 5 novel Photosystem I mRNA maturation factors validates this resource C_LIO_LIMTF1 and PMR1 identified as master regulators of photosynthesis C_LI

cell biology↗

bioRxiv2022

A Chloroplast Protein Atlas Reveals Novel Structures and Spatial Organization of Biosynthetic Pathways

Chloroplasts are eukaryotic photosynthetic organelles that drive the global carbon cycle. Despite their importance, our understanding of their protein composition, function, and spatial organization remains limited. Here, we determined the localizations of 1,032 candidate chloroplast proteins by using fluorescent protein tagging in the model alga Chlamydomonas reinhardtii. The localizations provide insights into the functions of hundreds of poorly-characterized proteins, including identifying novel components of nucleoids, plastoglobules, and the pyrenoid. We discovered and further characterized novel organizational features, including eleven chloroplast punctate structures, cytosolic crescent structures, and diverse unexpected spatial distributions of enzymes within the chloroplast. We observed widespread protein targeting to multiple organelles, identifying proteins that likely function in multiple compartments. We also used machine learning to predict the localizations of all Chlamydomonas proteins. The strains and localization atlas developed here will serve as a resource to enable studies of chloroplast architecture and functions. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/493820v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@19c871eorg.highwire.dtl.DTLVardef@16ac46corg.highwire.dtl.DTLVardef@859b7dorg.highwire.dtl.DTLVardef@1a3446e_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LI1,032 candidate chloroplast proteins localized by fluorescent tagging. C_LIO_LIThis protein atlas reveals novel chloroplast structures, functional regions, and components. C_LIO_LIPrevalent dual-organelle localization suggests extensive cross-compartment coordination. C_LIO_LIAtlas-trained machine learning predicts localizations of all C. reinhardtii proteins. C_LI

plant biology↗

bioRxiv2020

Systematic characterization of gene function in a photosynthetic organism

Photosynthetic organisms are essential for human life, yet most of their genes remain functionally uncharacterized. Single-celled photosynthetic model systems have the potential to accelerate our ability to connect genes to functions. Here, using a barcoded mutant library of the model eukaryotic alga Chlamydomonas reinhardtii, we determined the phenotypes of more than 58,000 mutants under more than 121 different environmental growth conditions and chemical treatments. 78% of genes are represented by at least one mutant that showed a phenotype, providing clues to the functions of thousands of genes. Mutant phenotypic profiles allow us to place known and previously uncharacterized genes into functional pathways such as DNA repair, photosynthesis, the CO2-concentrating mechanism, and ciliogenesis. We illustrate the value of this resource by validating novel phenotypes and gene functions, including the discovery of three novel components of a defense pathway that counteracts actin cytoskeleton inhibitors released by other organisms. The data also inform phenotype discovery in land plants: mutants in Arabidopsis thaliana genes exhibit similar phenotypes to those we observed in their Chlamydomonas homologs. We anticipate that this resource will guide the functional characterization of genes across the tree of life.

systems biology↗

bioRxiv2020

The Structural Basis of Rubisco Phase Separation in the Pyrenoid

Approximately one-third of global CO2 fixation occurs in a phase separated algal organelle called the pyrenoid. Existing data suggest that the pyrenoid forms by the phase-separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase-separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein EPYC1 (Essential Pyrenoid Component 1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly-spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of one of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation, and pyrenoid formation. Cryo-electron tomography supports a model where EPYC1 and Rubisco form a co-dependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

molecular biology↗

bioRxiv2020

Assembly of the algal CO2-fixing organelle, the pyrenoid, is guided by a Rubisco-binding motif

Approximately one-third of the Earths photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid, and how the organelles sub-compartments - membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath - are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we discovered that pyrenoid proteins share a sequence motif. We show that the motif is sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix-starch sheath interface suggests that the motif holds the pyrenoids three sub-compartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multi-layered phase-separated organelle. One Sentence SummaryA ubiquitous Rubisco-binding motif targets proteins to the pyrenoid and holds together the pyrenoids three sub-compartments.

cell biology↗

bioRxiv2018

A genome-wide algal mutant library reveals a global view of genes required for eukaryotic photosynthesis

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms that complement the use of multicellular plants by enabling higher-throughput studies. Here, we generated a genome-wide, indexed library of mapped insertion mutants for the flagship unicellular alga Chlamydomonas reinhardtii (Chlamydomonas hereafter). The 62,389 mutants in the library, covering 83% of nuclear, protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We leveraged this feature to perform a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase CPL3, showed it is important for accumulation of multiple photosynthetic protein complexes. Strikingly, 21 of the 43 highest-confidence genes are novel, opening new opportunities for advances in our understanding of this biogeochemically fundamental process. This library is the first genome-wide mapped mutant resource in any unicellular photosynthetic organism, and will accelerate the characterization of thousands of genes in algae, plants and animals.

genomics↗