Selective transport of fluorescent proteins into the phage nucleus.
Selective transport of fluorescent proteins into the phage nucleus.
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DOI：
10.1371/journal.pone.0251429
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发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Pogliano J
中科院分区：
文献类型：
--
作者：
Nguyen KT;Sugie J;Khanna K;Egan ME;Birkholz EA;Lee J;Beierschmitt C;Villa E;Pogliano J
在感染假单胞菌细胞后，巨噬菌体201Φ2-1、ΦPA3和ΦKZ组装成噬菌体核。病毒DNA被包裹在噬菌体编码的蛋白质外壳中，以及与DNA复制、重组和转录相关的蛋白质。参与代谢过程的核糖体和蛋白质被排除在细胞核之外。RNA合成发生在噬菌体细胞核内，信使RNA可能被运输到细胞质中进行翻译。新合成的蛋白质要么留在细胞质中，要么专门移位到细胞核中。在这些噬菌体感染系统中，控制选择性蛋白质分选和核输入的分子机制目前尚不清楚。为了深入了解这一过程，我们研究了五种报告荧光蛋白(GFP+、sfGFP、GFP mu1、mCherry、CFP)的定位。在感染ΦPA3或201Φ2-1期间，所有五种荧光蛋白都如预期的那样被排除在细胞核之外；然而，我们发现了ΦKZ核运输系统的异常。将自行表达的荧光蛋白GFP-mu1转运到Φ-KZ噬菌体细胞核中。我们鉴定了核靶向所需的GFP-mu1表面的氨基酸残基。将GFPmu1与任何蛋白质融合，包括通常驻留在细胞质中的蛋白质，导致融合进入细胞核。尽管转运机制尚不清楚，但我们证明GFP变1是一种有用的工具，可用于荧光标记和将蛋白质靶向ΦKZ噬菌体细胞核。
Upon infection of Pseudomonas cells, jumbo phages 201Φ2–1, ΦPA3, and ΦKZ assemble a phage nucleus. Viral DNA is enclosed within the phage-encoded proteinaceous shell along with proteins associated with DNA replication, recombination and transcription. Ribosomes and proteins involved in metabolic processes are excluded from the nucleus. RNA synthesis occurs inside the phage nucleus and messenger RNA is presumably transported into the cytoplasm to be translated. Newly synthesized proteins either remain in the cytoplasm or specifically translocate into the nucleus. The molecular mechanisms governing selective protein sorting and nuclear import in these phage infection systems are currently unclear. To gain insight into this process, we studied the localization of five reporter fluorescent proteins (GFP+, sfGFP, GFPmut1, mCherry, CFP). During infection with ΦPA3 or 201Φ2–1, all five fluorescent proteins were excluded from the nucleus as expected; however, we have discovered an anomaly with the ΦKZ nuclear transport system. The fluorescent protein GFPmut1, expressed by itself, was transported into the ΦKZ phage nucleus. We identified the amino acid residues on the surface of GFPmut1 required for nuclear targeting. Fusing GFPmut1 to any protein, including proteins that normally reside in the cytoplasm, resulted in transport of the fusion into the nucleus. Although the mechanism of transport is still unknown, we demonstrate that GFPmut1 is a useful tool that can be used for fluorescent labelling and targeting of proteins into the ΦKZ phage nucleus.
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