Like most major enzyme families, the M14 family of metallocarboxypeptidases (MCPs) contains a number of pseudoenzymes predicted to lack enzyme activity and with poorly characterized molecular function. The genome of the yeast Saccharomyces cerevisiae encodes one member of the M14 MCP family, a pseudoenzyme named Ecm14 proposed to function in the extracellular matrix. In order to better understand the function of such pseudoenzymes, we studied the structure and function of Ecm14 in S. cerevisiae. A phylogenetic analysis of Ecm14 in fungi found it to be conserved throughout the ascomycete phylum, with a group of related pseudoenzymes found in basidiomycetes. To investigate the structure and function of this conserved protein, His6-tagged Ecm14 was overexpressed in Sf9 cells and purified. The prodomain of Ecm14 was cleaved in vivo and in vitro by endopeptidases, suggesting an activation mechanism; however, no activity was detectable using standard carboxypeptidase substrates. In order to determine the function of Ecm14 using an unbiased screen, we undertook a synthetic lethal assay. Upon screening approximately 27,000 yeast colonies, twenty-two putative synthetic lethal clones were identified. Further analysis showed many to be synthetic lethal with auxotrophic marker genes and requiring multiple mutations, suggesting that there are few, if any, single S. cerevisiae genes that present synthetic lethal interactions with ecm14Δ. We show in this study that Ecm14, although lacking detectable enzyme activity, is a conserved carboxypeptidase-like protein that is secreted from cells and is processed to a mature form by the action of an endopeptidase. Our study and datasets from other recent large-scale screens suggest a role for Ecm14 in processes such as vesicle-mediated transport and aggregate invasion, a fungal process that has been selected against in modern laboratory strains of S. cerevisiae.

中文翻译：

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Ecm14，一种保守的真菌假肽酶的生化和遗传分析

像大多数主要的酶家族一样，M14家族的金属羧肽酶（MCPs）包含许多假酶，这些假酶预计缺乏酶活性，并且分子功能较差。酿酒酵母酵母的基因组编码M14 MCP家族的一个成员，该假酶名为Ecm14，拟在细胞外基质中发挥作用。为了更好地了解这种假酶的功能，我们研究了酿酒酵母中Ecm14的结构和功能。对真菌中Ecm14的系统发育分析发现，它在整个子囊动物门中都是保守的，在担子菌中发现了一组相关的假酶。为了研究这种保守蛋白的结构和功能，在Sf9细胞中过表达His6标签的Ecm14并进行纯化。Ecm14的前结构域被内肽酶在体内和体外裂解，提示其激活机制。但是，使用标准羧肽酶底物无法检测到活性。为了使用无偏屏幕确定Ecm14的功能，我们进行了合成致死分析。通过筛选大约27,000个酵母菌落，鉴定出22个推定的合成致死克隆。进一步的分析表明，许多具有营养缺陷型标记基因的合成致死性致死菌，需要多个突变，这表明，如果有的话，很少有单个酿酒酵母基因呈现与ecm14Δ的合成致死性相互作用。我们在这项研究中显示，Ecm14尽管缺乏可检测的酶活性，是一种保守的羧肽酶样蛋白，从细胞分泌，并通过内肽酶的作用加工成成熟形式。我们的研究和来自其他近期大规模筛选的数据集表明，Ecm14在囊泡介导的运输和聚集体入侵等过程中发挥了作用，而真菌过程已被酿酒酵母现代实验室菌株所针对。