Biology subjects

Ergun, S. L.

Publications and source records attributed to Ergun, S. L..

6 recordsLinked to original sources

bioRxiv2026

A bifunctional coiled-coil protein generates the membrane-within-condensate architecture of the CO2-fixing pyrenoid

How membranes are integrated into biomolecular condensates is a fundamental question in cell biology. In the algal pyrenoid, an organelle responsible for one-third of global carbon fixation, CO2-delivering thylakoid membranes must penetrate a phase-separated condensate of the CO2-fixing enzyme Rubisco, but the mechanism governing membrane recruitment into the condensate remains unknown. Here, we demonstrate that the Chlamydomonas reinhardtii protein MITH1 acts as a molecular anchor that brings membrane into the pyrenoid condensate. MITH1 dimerizes into an extended coiled coil with an N-terminal amphipathic helix that binds thylakoid membrane. The coiled coil contains multiple novel binding sites for the Rubisco large subunit, allowing the condensate to wet onto the membrane. The coiled coil extends away from the membrane and promotes membrane organization within the condensate. These findings solve a longstanding mechanistic question in pyrenoid biogenesis and reveal general principles for how membranes are integrated into biological condensates.

molecular biology↗

bioRxiv2025

Kinase KEY1 controls pyrenoid condensate size throughout the cell cycle by disrupting phase separation interactions

Biomolecular condensates spatially organize cellular functions, but the regulation of their size, number, dissolution, and re-condensation is poorly understood. The pyrenoid, an algal biomolecular condensate that mediates one-third of global CO2 fixation, typically exists as one large condensate per chloroplast, but during cell division it transiently dissolves and reconfigures into multiple smaller condensates. Here, we identify a kinase, KEY1, in the model alga Chlamydomonas reinhardtii that regulates pyrenoid condensate size and number dynamics throughout the cell cycle and is necessary for normal pyrenoid function and growth. Unlike wild type, key1 mutant cells have multiple smaller condensates throughout the cell cycle that fail to dissolve during cell division. We show that KEY1 localizes to the condensates and promotes their dissolution by disrupting interactions between their core constituents, the CO2-fixing enzyme Rubisco and its linker protein EPYC1, through EPYC1 phosphorylation. We develop a biophysical model that recapitulates KEY1-mediated condensate size and number regulation and suggests a mechanism for controlling condensate position. These data provide a foundation for the mechanistic understanding of the regulation of size, number, position, and dissolution in pyrenoids and other biomolecular condensates.

cell biology↗

bioRxiv2024

Proteomic analysis of the pyrenoid-traversing membranes of Chlamydomonas reinhardtii reveals novel components

O_LIPyrenoids are algal CO2-fixing organelles that mediate approximately one-third of global carbon fixation. Most pyrenoids are traversed by membranes that are thought to supply them with concentrated CO2. Despite the critical nature of these membranes for pyrenoid function, they are poorly understood, with few protein components known in any species. C_LIO_LIHere, we identify protein components of the pyrenoid-traversing membranes from the leading model alga, Chlamydomonas reinhardtii, by affinity purification and mass spectrometry of membrane fragments. Our proteome includes previously-known proteins as well as novel candidates. C_LIO_LIWe further characterize two of the novel pyrenoid-traversing membrane-resident proteins: Cre10.g452250, which we name Pyrenoid Membrane Enriched 1 (PME1), and Cre02.g143550, also known as Low-CO2-Induced 16 (LCI16). We confirm the pyrenoid-traversing membrane localization of LCI16 and observe that PME1 and LCI16 physically interact. We find that neither protein is required for normal membrane morphology or growth under CO2-limiting conditions, but that both mutants show a similar proteomic profile to those of established pyrenoid mutants. C_LIO_LITaken together, our study identifies the proteome of the pyrenoid-traversing membranes and initiates the characterization of a novel pyrenoid-traversing membrane complex, building toward a mechanistic understanding of the pyrenoid. C_LI

molecular biology↗

bioRxiv2024

Biogenesis, engineering and function of membranes in the CO2-fixing pyrenoid

Summary ParagraphApproximately one-third of global CO2 assimilation is performed by the pyrenoid1, a liquid-like organelle found in most algae and some plants2. Specialized membranes are hypothesized to drive CO2 assimilation in the pyrenoid by delivering concentrated CO23,4, but their biogenesis and function have not been experimentally characterized. Here, we show that homologous proteins SAGA1 and MITH1 mediate the biogenesis of the pyrenoid membrane tubules in the model alga Chlamydomonas reinhardtii and are sufficient to reconstitute pyrenoid-traversing membranes in a heterologous system, the plant Arabidopsis thaliana. SAGA1 localizes to the regions where thylakoid membranes transition into tubules and is necessary to initiate tubule formation. MITH1 localizes to the tubules and is necessary for their extension through the pyrenoid. Tubule-deficient mutants exhibit growth defects under CO2-limiting conditions, providing evidence for the function of membrane tubules in CO2 delivery to the pyrenoid. Furthermore, these mutants form multiple aberrant condensates of pyrenoid matrix, indicating that a normal tubule network promotes the coalescence of a single pyrenoid. The reconstitution of pyrenoid-traversing membranes in a plant represents a key milestone toward engineering a functional pyrenoid into crops for improving crop yields. More broadly, our study demonstrates the functional importance of pyrenoid membranes, identifies key biogenesis factors, and paves the way for the molecular characterization of pyrenoid membranes across the tree of life.

molecular biology↗

bioRxiv2023

Blocking oligomerization is the most viable strategy to inhibit STING

The anti-viral and anti-cancer STING innate immune pathway can exacerbate autoimmune and neurodegenerative diseases when aberrantly activated, emphasizing a key unmet need for STING pathway antagonists. However, no such inhibitors have advanced to the clinic because it remains unclear which mechanistic step(s) of human STING activation are crucial for potent and context-independent inhibition of downstream signaling. Here, we report that C91 palmitoylation, the mechanistic target of a potent tool compound, is not universally necessary for human STING signaling, making it a poor target for drug development. Instead, we discover that evolutionarily conserved C64 is basally palmitoylated and is crucial for preventing unproductive STING oligomerization in the absence of cGAMP stimulation. The effects of palmitoylation at C64 and C91 converge on the control of intra-dimer disulfide bond formation at C148. Importantly, we show for the first time that signaling-competent STING oligomers are composed of a mixture of two species: disulfide-linked STING dimers that stabilize the oligomer, and reduced STING dimers that are phosphorylated to actuate interferon signaling. Given this complex landscape and cell type specificity of palmitoylation modifications, we conclude that robust STING inhibitors must directly inhibit the oligomerization process. Taking inspiration from STINGs natural autoinhibitory mechanism, we identified an eight amino acid peptide that binds a defined pocket at the inter-dimer oligomerization interface as a proof-of-concept human STING inhibitor, setting the stage for future therapeutic development. SummaryWe report that functional STING oligomers require palmitoylation at cysteine 64 and some proportion of reduced dimers, and define the site of autoinhibition that can be targeted to disrupt STING oligomerization and activity.

biochemistry↗

bioRxiv2019

STING polymer structure reveals mechanisms for activation, hyperactivation, and inhibition

How the central innate immune protein, STING, is activated by its ligands remains unknown. Here, using structural biology and biochemistry, we report that the metazoan second messenger 23-cGAMP induces closing of the human STING homodimer and release of the STING C-terminal tail, which exposes a polymerization interface on the STING dimer and leads to the formation of disulfide-linked polymers via cysteine residue 148. Disease-causing hyperactive STING mutations either flank C148 and depend on disulfide formation or reside in the C-terminal tail binding site and cause constitutive C-terminal tail release and polymerization. Finally, bacterial cyclic-di-GMP induces an alternative active STING conformation, activates STING in a cooperative manner, and acts as a partial antagonist of 23-cGAMP signaling. Our insights explain the tight control of STING signaling given varying background activation signals and provide a novel therapeutic hypothesis for autoimmune syndrome treatment.

biochemistry↗