BACKGROUND & AIMS: :The yeast (Saccharomyces cerevisiae) Snf7 family consists of six highly charged, coiled-coil-forming proteins involved in multivesicular body (MVB) formation. Although all proteins perform a common function at late endosomes, individual mutants also show distinct phenotypes. This suggests that Snf7 homologues have additional functions separate from their role in MVB formation. In this report, we explored the molecular basis for the sucrose-nonfermenting phenotype of snf7 mutants. Our Northern blotting experiments provide evidence that Snf7 is involved in the regulation of SUC2 transcription. The Snf7-dependent regulation of SUC2 transcription does not appear to involve the transcription factor Mig1, since Mig1 phosphorylation after glucose derepression was not affected in a Deltasnf7 mutant. Instead, we show that Snf7 influences SUC2 expression by regulating the level of the transcription factor Nrg1. Snf7 exerts its effects on Nrg1 levels through activation of the transcription factor Rim101, which is part of the yeast alkaline response pathway ("Rim101 pathway"). This is supported by the findings that deletion of RIM101 or overexpression of NRG1 from the ADH1 promoter leads to the same SUC2 expression level as deletion of SNF7. In addition, deletion of other components of the Rim101 pathway, like RIM13 and RIM20, led to the same growth phenotype on raffinose media as deletion of SNF7. Furthermore, Snf7 turned out to be dispensable for SUC2 expression in an NRG1 deletion background. Thus, the effects of Snf7 on SUC2 expression can be completely accounted for by its effect on Nrg1 levels.

背景与目标： ：酵母（Saccharomyces cerevisiae）Snf7家族由六种高度带电荷的卷曲螺旋形成蛋白组成，这些蛋白参与多囊泡体（MVB）的形成。尽管所有蛋白质都在晚期内体中发挥共同的功能，但单个突变体也显示出不同的表型。这表明Snf7同源物除了在MVB形成中的作用外，还具有其他功能。在本报告中，我们探讨了snf7突变体蔗糖非发酵表型的分子基础。我们的RNA印迹实验提供了Snf7参与SUC2转录调控的证据。 SUC2转录的Snf7依赖性调节似乎不涉及转录因子Mig1，因为在Deltasnf7突变体中葡萄糖抑制后的Mig1磷酸化不受影响。相反，我们显示Snf7通过调节转录因子Nrg1的水平影响SUC2表达。 Snf7通过激活转录因子Rim101来发挥其对Nrg1水平的影响，Rim101是酵母碱性反应途径（“ Rim101途径”）的一部分。这由以下发现支持：RIM101的缺失或ADH1启动子中NRG1的过表达导致与SNF7缺失相同的SUC2表达水平。此外，Rim101途径的其他成分（如RIM13和RIM20）的缺失导致棉子糖培养基上的生长表型与SNF7的缺失相同。此外，事实证明，Snf7对于NRG1缺失背景中的SUC2表达而言是可有可无的。因此，Snf7对SUC2表达的影响可以完全通过其对Nrg1水平的影响来解释。

BACKGROUND & AIMS: :Candida albicans is an opportunistic pathogen that colonizes diverse mucosal niches with distinct environmental characteristics. To adapt to these different sites, C. albicans must activate and attenuate a variety of signal transduction pathways. A mechanism of signal attenuation is through receptor endocytosis and subsequent vacuolar degradation, which requires the endosomal sorting complex required for transport (ESCRT) pathway. This pathway comprises several polyprotein complexes (ESCRT-0, -I, -II, -III, and -DS) that are sequentially recruited to the endosomal membrane. The ESCRT pathway also activates the Rim101 transcription factor, which governs expression of genes required for virulence. Here, we tested the hypothesis that the ESCRT pathway plays a Rim101-independent role(s) in pathogenesis. We generated deletion mutants in each ESCRT complex and determined that ESCRT-I, -II, and -III are required for Rim101 activation but that ESCRT-0 and ESCRT-DS are not. We found that the ESCRT-0 member Vps27 and ESCRT-DS components are required to promote epithelial cell damage and, using a murine model of oral candidiasis, found that the vps27Delta/Delta mutant had a decreased fungal burden compared to that of the wild type. We found that a high-dose inoculum can compensate for fungal burden defects but that mice colonized with the vps27Delta/Delta strain exhibit less morbidity than do mice infected with the wild-type strain. These results demonstrate that the ESCRT pathway has Rim101-independent functions for C. albicans virulence.

背景与目标： ：白色念珠菌是一种机会病原体，它定居在具有独特环境特征的各种粘膜壁ni中。为了适应这些不同的位置，白色念珠菌必须激活并减弱各种信号转导途径。信号衰减的机制是通过受体内吞作用和随后的液泡降解，这需要转运所需的内体分选复合物（ESCRT）途径。该途径包含依次募集到内体膜的几种多蛋白复合物（ESCRT-0，-I，-II，-III和-DS）。 ESCRT途径还激活Rim101转录因子，该因子控制毒力所需基因的表达。在这里，我们测试了ESCRT途径在发病机制中起Rim101独立作用的假设。我们在每个ESCRT复合物中生成了缺失突变体，并确定ESCRT-1，-II和-III是激活Rim101所必需的，而ESCRT-0和ESCRT-DS不是必需的。我们发现ESCRT-0成员Vps27和ESCRT-DS成分是促进上皮细胞损伤所必需的，并且使用口腔念珠菌病的鼠模型，发现vps27Delta / Delta突变体与野生型相比具有降低的真菌负担。我们发现高剂量接种物可以补偿真菌负荷缺陷，但是定居于vps27Delta / Delta菌株的小鼠的发病率要低于感染野生型菌株的小鼠。这些结果表明，ESCRT途径对白色念珠菌的毒力具有Rim101独立的功能。

BACKGROUND & AIMS: :Ambient pH signalling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Insertional mutagenesis in the yeast Yarrowia lipolytica identified two components of the endosome-associated ESCRT-I complex involved in multivesicular body (MVB) vesicle formation, YlVps28p and YlVps23p. They were shown to be required at alkaline pH, like Rim factors, for transcriptional activation of alkaline-induced genes and repression of acid-induced genes. The constitutively active YlRIM101-1119 allele, which suppresses the pH-signalling defects of Ylrim mutations, also suppresses Ylvps defects in pH response, but not in endocytosis. The contribution of the ESCRT-III component Snf7p could not be assessed due to the essential nature of this component in Y. lipolytica. Unlike Rim factors, YlVps4p, a component of the MVB pathway acting downstream from ESCRT complexes, seems not to be required for the alkaline response. In Y. lipolytica, all vps mutations including those affecting YlVPS4, affected growth at acidic pH, a feature not exhibited by Ylrim mutations. These results suggest that Rim and Vps pathways cooperate in ambient pH signalling and that this relation is conserved across the full range of hemiascomycetous yeasts.

背景与目标： ：环境pH信号涉及分别在子囊酵母或丝状真菌中级联的保守的Rim或Pal产物。酵母解脂耶氏酵母中的插入诱变鉴定了与多囊体（MVB）囊泡形成有关的内体相关的ESCRT-1复合物的两个组分，Y1Vps28p和Y1Vps23p。已显示出它们需要像Rim因子一样在碱性pH值下对碱性诱导的基因进行转录激活并抑制酸性诱导的基因。抑制Ylrim突变的pH信号缺陷的组成型活性Y1RIM101-1119等位基因，也抑制pH响应中的Ylvps缺陷，但不抑制内吞作用。由于解脂耶氏酵母中该成分的本质，因此无法评估ESCRT-III成分Snf7p的贡献。与Rim因子不同，Y1Vps4p是在ESCRT复合物中下游起作用的MVB途径的一个组成部分，似乎对于碱性反应不是必需的。在解脂耶氏酵母中，所有vps突变（包括那些影响Y1VPS4的突变）都在酸性pH下影响了生长，这是Ylrim突变所没有表现出的特征。这些结果表明，Rim和Vps途径在环境pH信号转导中协同作用，并且这种关系在整个半糖酵母中都得到了保留。

BACKGROUND & AIMS: :Saccharomyces cerevisiae cells activate the Rim101 pathway to adapt to alkaline and salt stresses. On activation of this pathway, the transcription factor Rim101 undergoes proteolytic activation and regulates the expression of responsive genes. We found Rim101 to be a short-lived protein with a half-life of approximately 15 min. Its rapid turnover was supposedly mediated by the ubiquitin-proteasome system. Excess accumulation of the processed active Rim101 through its over-expression conferred tolerance to both alkaline and salt stresses in yeast cells; in contrast, it had detrimental effects under cadmium stress condition. Cadmium ion inhibited proteolytic activation of Rim101, implying reciprocal interaction between the Rim101 pathway and cadmium stress. Our results showed yeast cells to be equipped with two protective systems to prevent overaccumulation of the processed active Rim101; Rim101 processing is inhibited when Rim101 level is high, and turnover of processed Rim101 is accelerated when it is abundant. Collectively, the results confirmed the flexible aspect of stress response in yeast cell; the cells not only prevent excess activation of one stress-responsive pathway but also facilitate its attenuation to cope with other environmental stresses.

背景与目标： ：酿酒酵母细胞激活Rim101途径以适应碱性和盐胁迫。激活该途径后，转录因子Rim101进行蛋白水解激活并调节反应性基因的表达。我们发现Rim101是一种短暂的蛋白质，半衰期约为15分钟。据称它的快速周转是由泛素-蛋白酶体系统介导的。加工过的活性Rim101的过量表达导致的过量积累赋予了其对酵母细胞中碱和盐胁迫的耐受性；相反，它在镉胁迫条件下具有有害作用。镉离子抑制Rim101的蛋白水解激活，这暗示Rim101途径与镉胁迫之间的相互作用。我们的结果表明，酵母细胞配备了两个保护系统，以防止加工过的活性Rim101过度积聚。当Rim101的水平高时，Rim101的处理被禁止，而当Rim101的水平高时，则处理的Rim101的营业额被加速。总体而言，结果证实了酵母细胞中应激反应的灵活方面。这些细胞不仅阻止了一种应激反应途径的过度激活，而且还促进了其衰减以应对其他环境胁迫。

BACKGROUND & AIMS: :Growth and differentiation of Candida albicans over a broad pH range underlie its ability to infect an array of tissues in susceptible hosts. We identified C. albicans RIM101, RIM20, and RIM8 based on their homology to components of the one known fungal pH response pathway. PCR product-disruption mutations in each gene cause defects in three responses to alkaline pH: filamentation, induction of PRA1 and PHR1, and repression of PHR2. We find that RIM101 itself is an alkaline-induced gene that also depends on Rim20p and Rim8p for induction. Two observations indicate that a novel pH response pathway also exists. First, PHR2 becomes an alkaline-induced gene in the absence of Rim101p, Rim20p, or Rim8p. Second, we created strains in which Rim101p activity is independent of Rim20p and Rim8p; in these strains, filamentation remains pH dependent. Thus, pH governs gene expression and cellular differentiation in C. albicans through both RIM101-dependent and RIM101-independent pathways.

背景与目标： ：白色念珠菌在广泛的pH范围内的生长和分化是其感染易感宿主的一系列组织的能力的基础。我们基于白色念珠菌RIM101，RIM20和RIM8与一种已知真菌pH响应途径的组成部分的同源性进行了鉴定。每个基因中的PCR产物破坏突变都会导致对碱性pH值的三种反应出现缺陷：丝状化，PRA1和PHR1的诱导以及PHR2的抑制。我们发现RIM101本身是碱性诱导的基因，其诱导也依赖于Rim20p和Rim8p。两项观察结果表明，还存在一种新型的pH响应途径。首先，在缺少Rim101p，Rim20p或Rim8p的情况下，PHR2成为碱性诱导的基因。其次，我们创建了其中Rim101p活性独立于Rim20p和Rim8p的菌株。在这些菌株中，丝状化仍然依赖于pH。因此，pH通过RIM101依赖性和RIM101依赖性途径控制白色念珠菌的基因表达和细胞分化。

BACKGROUND & AIMS: :Morphological development of the fungal pathogen Candida albicans is profoundly affected by ambient pH. Acidic pH restricts growth to the yeast form, whereas neutral pH permits development of the filamentous form. Superimposed on the pH restriction is a temperature requirement of approximately 37 degrees C for filamentation. The role of pH in development was investigated by selecting revertants of phr2Delta mutants that had gained the ability to grow at acid pH. The extragenic suppressors in two independent revertants were identified as nonsense mutations in the pH response regulator RIM101 (PRR2) that resulted in a carboxy-terminal truncation of the open reading frame. These dominant active alleles conferred the ability to filament at acidic pH, to express PHR1, an alkaline-expressed gene, at acidic pH, and to repress the acid-expressed gene PHR2. It was also observed that both the wild-type and mutant alleles could act as multicopy suppressors of the temperature restriction on filamentation, allowing extensive filamentation at 29 degrees C. The ability of the activated alleles to promote filamentation was dependent upon the developmental regulator EFG1. The results suggest that RIM101 is responsible for the pH dependence of hyphal development.

背景与目标： ：真菌病原体白色念珠菌的形态发育受到环境pH的深刻影响。酸性pH限制了酵母形式的生长，而中性pH允许形成丝状形式。在pH限制上叠加了长丝所需的温度约为37摄氏度。通过选择具有在酸性pH条件下生长能力的phr2Delta突变体的回复株，研究了pH在发育中的作用。在两个独立回复子中的外源抑制剂被鉴定为pH响应调节剂RIM101（PRR2）中的无意义突变，导致开放阅读框的羧基末端被截断。这些占优势的活性等位基因赋予了在酸性pH下发丝，表达PHR1（在碱性pH下表达碱性表达的基因）和抑制酸性表达基因PHR2的能力。还观察到，野生型和突变型等位基因均可以充当丝状体温度限制的多拷贝抑制剂，从而允许在29摄氏度下广泛地丝状化。活化的等位基因促进丝状体形成的能力取决于发育调节剂EFG1。结果表明RIM101负责菌丝发育的pH依赖性。

BACKGROUND & AIMS: :Ambient pH signaling involves a cascade of conserved Rim or Pal products in ascomycetous yeasts or filamentous fungi, respectively. Recent evidences in the fungi Aspergillus nidulans, Saccharomyces cerevisiae, Yarrowia lipolytica, and Candida albicans suggested that components of endosomal sorting complexes required for transport (ESCRT) involved in endocytic trafficking were needed for signal transduction along the Rim pathway. In this study, we confirm these findings with C. albicans and show that Vps28p (ESCRT-I) and Vps32p/Snf7p (ESCRT-III) are required for the transcriptional regulation of known targets of the Rim pathway, such as the PHR1 and PHR2 genes encoding cell surface proteins, which are expressed at alkaline and acidic pH, respectively. We additionally show that deletion of these two VPS genes, particularly VPS32, has a more drastic effect than a RIM101 deletion on growth at alkaline pH and that this effect is only partially suppressed by expression of a constitutively active form of Rim101p. Finally, in an in vivo mouse model, both vps null mutants were significantly less virulent than a rim101 mutant, suggesting that VPS28 and VPS32 gene products affect virulence both through Rim-dependent and Rim-independent pathways.

背景与目标： ：环境pH信号涉及分别在子囊酵母或丝状真菌中级联的保守的Rim或Pal产物。在真菌曲霉，酿酒酵母，解脂耶氏酵母和白色念珠菌中的最新证据表明，参与内吞运输的运输所需的内体分选复合物（ESCRT）的成分需要沿Rim途径进行信号转导。在这项研究中，我们用白色念珠菌证实了这些发现，并显示了Rps途径的已知靶标（例如PHR1和PHR2）的转录调控需要Vps28p（ESCRT-I）和Vps32p / Snf7p（ESCRT-III）基因编码细胞表面蛋白，分别在碱性和酸性pH下表达。我们还显示，在碱性pH值下，这两个VPS基因（尤其是VPS32）的缺失比RIM101缺失对生长的影响更大，并且仅通过Rim101p的组成型活性形式的表达来部分抑制这种作用。最后，在体内小鼠模型中，两个vps null突变体的毒性都比rim101突变体低得多，这表明VPS28和VPS32基因产物通过Rim依赖性和Rim依赖性途径影响毒力。

BACKGROUND & AIMS: :Good's syndrome (GS) is a rare acquired combined T- and B-cell immunodeficiency accompanying thymoma. This report concerns a case of a 57-year-old man with GS manifesting intractable opportunistic infections and hyperkeratotic lichen planus. He had a past history of extended thymectomy for removal of thymoma. He consulted us about scaly and exudative intractable erythematous plaque on his right forearm. The histology was compatible with phlegmon coexisting with lichen planus. Laboratory examination results indicated hypogammaglobulinemia accompanied by complete absence of B cells, which is consistent with GS. Combined treatment with immunoglobulin replacement and administration of antibiotics and antifungal drugs was effective for the phlegmon and overlying fungal infection. The patient also presented with hyperkeratotic lichen planus on both knees and the right elbow, suggesting that intractable opportunistic infection and lichen planus may be associated with GS.

背景与目标： ：Good's综合征（GS）是伴有胸腺瘤的一种罕见的获得性T细胞和B细胞免疫缺陷综合症。该报告涉及一例GS的57岁男子，该男子表现出顽固的机会性感染和角化过度的扁平苔藓。他有去除胸腺瘤的扩大胸腺切除术的历史。他向我们咨询了右前臂上的鳞状和渗出性难治性红斑。组织学与扁平苔藓并存的痰菌相容。实验室检查结果表明低血球蛋白血症伴有B细胞的完全缺失，这与GS一致。结合免疫球蛋白替代疗法和抗生素及抗真菌药物的联合治疗对痰菌和上层真菌感染均有效。该患者在膝盖和右肘上均出现角化过度的扁平苔藓，提示难治性机会性感染和扁平苔藓可能与GS有关。

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BACKGROUND & AIMS: :Modulating composition and shape of biological membranes is an emerging mode of regulation of cellular processes. We investigated the global effects that such perturbations have on a model eukaryotic cell. Phospholipases A(2) (PLA(2)s), enzymes that cleave one fatty acid molecule from membrane phospholipids, exert their biological activities through affecting both membrane composition and shape. We have conducted a genome-wide analysis of cellular effects of a PLA(2) in the yeast Saccharomyces cerevisiae as a model system. We demonstrate functional genetic and biochemical interactions between PLA(2) activity and the Rim101 signaling pathway in S. cerevisiae. Our results suggest that the composition and/or the shape of the endosomal membrane affect the Rim101 pathway. We describe a genetically and functionally related network, consisting of components of the Rim101 pathway and the prefoldin, retromer and SWR1 complexes, and predict its functional relation to PLA(2) activity in a model eukaryotic cell. This study provides a list of the players involved in the global response to changes in membrane composition and shape in a model eukaryotic cell, and further studies are needed to understand the precise molecular mechanisms connecting them.

背景与目标： ：调节生物膜的组成和形状是调节细胞过程的一种新兴模式。我们研究了这种扰动对模型真核细胞的整体影响。磷脂酶A（2）（PLA（2）s）是一种从膜磷脂上切割一个脂肪酸分子的酶，通过影响膜的组成和形状来发挥其生物学活性。我们已经进行了全基因组范围内的酿酒酵母作为模型系统的PLA（2）的细胞作用的分析。我们展示了功能基因和生化相互作用之间的PLA（2）活动和啤酒酵母Rim101信号通路。我们的结果表明，内体膜的组成和/或形状会影响Rim101途径。我们描述了一个遗传和功能相关的网络，由Rim101途径和prefoldin，Retromer和SWR1复合物的组成组成，并预测其对模型真核细胞中PLA（2）活性的功能关系。这项研究提供了参与对真核细胞模型中膜组成和形状变化的整体反应的参与者的清单，还需要进一步的研究来了解连接它们的精确分子机制。