Yeast filamentation signaling is connected to a specific substrate translocation mechanism of the Mep2 transceptor
Yeast filamentation signaling is connected to a specific substrate translocation mechanism of the Mep2 transceptor
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DOI：
10.1371/journal.pgen.1008634
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发表时间：
2020-02-01
期刊：
影响因子：
4.5
通讯作者：
Boeckstaens, Melanie
中科院分区：
文献类型：
--
作者：
Brito, Ana Sofia;Neuhaeuser, Benjamin;Boeckstaens, Melanie
从酵母到丝状生长形式的二态转变允许细胞探索它们的环境以获得更合适的生态位，并且通常对致病真菌的毒力至关重要。与它们的Mep 1/3旁系同源物相反，保守的Mep-Rh-Rh家族的真菌Mep 2型铵转运蛋白已被分配了触发铵缺乏反应的信号所需的额外受体作用。在这里，遗传，动力学和结构-功能分析被用来阐明酿酒酵母Mep 2的信号转导作用。我们发现，缺乏C-末端尾的Mep 2变体保存诱导表达的能力，揭示了信号传导可以在没有推定的合作伙伴与蛋白质的最大胞质结构域的排他性结合的情况下进行。我们的数据支持，表达信号需要伴随底物易位通过孔跨越Mep 2的疏水核心的构象变化。pHluorin报告基因测定表明，Mep 2和非信号Mep 1的转运活性不同地影响体内酵母胞质pH，并且独特的孔变体Mep 2（H194 E）具有明显的转运和信号功能解偶联，获得了增强的酸化能力。在非洲爪蟾卵母细胞的功能特性表明，Mep 2介导电中性底物易位，而Mep 1进行产电运输。我们的研究结果强调了Mep 2依赖性的诱导与其特定的转运机制有关，表明pH在信号介导中的作用。最后，我们表明，信号传导过程是保守的Mep 2蛋白从人类病原体Candida albicans.Author摘要真菌Mep 2型铵转运蛋白的保守的Mep-Rh-Rh家族，包括人类恒河猴因子是特别需要的，以允许在响应于铵限制reproduction。因此，这些蛋白质被分配受体的作用，而信号转导的基本机制仍然知之甚少。随后提出了“转运体”性质，涉及从真菌到人类的真核生物中所有种类的微量和大量营养素的转运体。然而，由于难以获得转运和受体功能完全解偶联的变体，因此证明其存在仍然具有挑战性。我们的数据质疑酿酒酵母Mep 2的C-末端末端参与信号介导，从而导致表达。如果存在信号传导伴侣，它们也应该与胞质环和/或膜嵌入结构域结合。Mep 2的能力，使双折射是密切交织在一起的机制，通过蛋白质的疏水核心的孔的底物易位。在非洲爪蟾卵母细胞中，非信号Mep 1的运输活性是产电的，而Mep 2是电中性的，后者可能转运弱碱NH3，但不是NH 4+识别和去旋后释放的质子。我们建议，给定的后果的Mep 2特定的运输过程中，如细胞内的pH值的修改，可能是潜在的原因，确保Mep 2型蛋白质的抑制信号。
The dimorphic transition from the yeast to the filamentous form of growth allows cells to explore their environment for more suitable niches and is often crucial for the virulence of pathogenic fungi. In contrast to their Mep1/3 paralogues, fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family have been assigned an additional receptor role required to trigger the filamentation signal in response to ammonium scarcity. Here, genetic, kinetic and structure-function analyses were used to shed light on the poorly characterized signaling role of Saccharomyces cerevisiae Mep2. We show that Mep2 variants lacking the C-terminal tail conserve the ability to induce filamentation, revealing that signaling can proceed in the absence of exclusive binding of a putative partner to the largest cytosolic domain of the protein. Our data support that filamentation signaling requires the conformational changes accompanying substrate translocation through the pore crossing the hydrophobic core of Mep2. pHluorin reporter assays show that the transport activity of Mep2 and of non-signaling Mep1 differently affect yeast cytosolic pH in vivo, and that the unique pore variant Mep2(H194E), with apparent uncoupling of transport and signaling functions, acquires increased ability of acidification. Functional characterization in Xenopus oocytes reveals that Mep2 mediates electroneutral substrate translocation while Mep1 performs electrogenic transport. Our findings highlight that the Mep2-dependent filamentation induction is connected to its specific transport mechanism, suggesting a role of pH in signal mediation. Finally, we show that the signaling process is conserved for the Mep2 protein from the human pathogen Candida albicans.Author summary Fungal Mep2-type ammonium transport proteins of the conserved Mep-Amt-Rh family that includes human Rhesus factors are specifically required to allow filamentation in response to ammonium limitation. These proteins were therefore assigned a receptor role while the underlying mechanism of signal transduction remains poorly understood. The "transceptor" property has subsequently been proposed to concern transporters of all kind of micro- and macro- nutrients in eukaryotes, from fungi to human. However, bringing the firm demonstration of their existence remains challenging as variants with full uncoupling of transport and receptor functions are difficult to obtain. Our data question the involvement of the C-terminal extremity of Saccharomyces cerevisiae Mep2 in the signal mediation leading to filamentation. If signaling partners exist, they should also bind to cytosolic loops and/or membrane-embedded domains. The capacity of Mep2 to enable filamentation is closely intertwined to the mechanism of substrate translocation through the pore of the hydrophobic core of the protein. In Xenopus oocytes, the transport activity of non-signaling Mep1 is electrogenic while it is electroneutral for Mep2, the latter likely translocating the weak base NH3, but not the proton released after NH4+ recognition and depronotation. We propose that given consequences of a Mep2-specific transport process, such as an intracellular pH modification, could be the underlying cause of the filamentation signal ensured by Mep2-type proteins.