CyanoTag: Discovery of protein function facilitated by high-throughput endogenous tagging in a photosynthetic prokaryote
CyanoTag: Discovery of protein function facilitated by high-throughput endogenous tagging in a photosynthetic prokaryote
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DOI：
10.1101/2024.02.28.582475
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发表时间：
2024-02
期刊：
影响因子：
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通讯作者：
Abigail J. Perrin;Matthew Dowson;A. Dowle;Grant Calder;Victoria J. Springthorpe;Guoyan Zhao;L. Mackinder
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文献类型：
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作者：
Abigail J. Perrin;Matthew Dowson;A. Dowle;Grant Calder;Victoria J. Springthorpe;Guoyan Zhao;L. Mackinder
尽管它们对水生生态系统、全球碳循环和可持续生物工业的令人兴奋的潜力至关重要，但光合原核生物的基因组仍然包含大量未表征的蛋白质编码基因。在这里，我们提出了一个高通量的方法无瘢痕内源性荧光蛋白标记的模式蓝藻细长聚球藻PCC 7942。从400个靶标中，我们成功地内源性标记了超过330种蛋白质，对应于>10%的蛋白质组。我们演示了如何在规模上使用这种资源，以确定亚细胞定位，跟踪相对蛋白质丰度和阐明蛋白质-蛋白质相互作用网络。我们的数据为从光合作用到细胞分裂的各种过程提供了生物学见解。特别令人感兴趣的是，我们的“CyanoTag”线使我们能够在真实的时间内可视化卡尔文循环蛋白Prk和Gap 2在光线消失后的几秒钟内快速凝结，有效地“关闭”黑暗中的光合作用。这些见解，CyanoTag细胞系，相关数据和优化方法旨在作为一种资源共享，以促进与蓝藻相关的进一步发现，更广泛地涉及所有光合生命。
Despite their fundamental importance to aquatic ecosystems, global carbon cycling and exciting potential in sustainable bioindustries, the genomes of photosynthetic prokaryotes still contain large numbers of uncharacterised protein-coding genes. Here we present a high-throughput approach for scarless endogenous fluorescent protein tagging in the model cyanobacterium Synechococcus elongatus PCC7942. From 400 targets we successfully endogenously tag over 330 proteins corresponding to >10% of the proteome. We demonstrate how this resource can be used at scale to determine subcellular localisation, track relative protein abundances and to elucidate protein-protein interaction networks. Our data has provided biological insights into a diverse range of processes - from photosynthesis to cell division. Of particular interest, our ‘CyanoTag’ lines enabled us to visualise in real time the rapid condensation of Calvin Cycle proteins Prk and Gap2 within seconds of withdrawal of light, effectively ‘switching off’ photosynthesis in the dark. These insights, CyanoTag cell lines, associated data and optimised methods are intended to be shared as a resource to facilitate further discoveries relevant to cyanobacteria and more broadly to all photosynthetic life.