Localization of autophagy-related proteins in yeast using a versatile plasmid-based resource of fluorescent protein fusions
Localization of autophagy-related proteins in yeast using a versatile plasmid-based resource of fluorescent protein fusions
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DOI：
10.4161/auto.6308
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发表时间：
2008-08-16
期刊：
影响因子：
13.3
通讯作者：
Kumar, Anuj
中科院分区：
文献类型：
--
作者：
Ma, Jun;Bharucha, Nike;Kumar, Anuj
以质粒为基础的荧光蛋白融合集合是有价值的和通用的资源，有助于在多个遗传背景下对蛋白质定位的系统研究。然而，目前很少有这样的收集用于分析蛋白质在任何生物体中的定位。为了解决这一不足，我们在这里提出了一套基于质粒的资源，用于分析发芽酵母中的蛋白质定位。具体地说，我们构建了一套低拷贝的目的载体，用于基于重组的酵母基因的克隆作为荧光蛋白融合。我们克隆了一组编码激酶、转录因子和信号蛋白的384个酵母菌基因，作为“重组就绪”盒；通过Gateway克隆，这些带有天然启动子的基因可以很容易地导入到上述目的载体中，产生羧基末端融合成荧光蛋白。利用这些试剂，我们构建了276个编码黄色荧光蛋白(VYFP)的羧基末端融合基因的亚集。这个集合包括14个自噬相关(ATG)基因，我们在雷帕霉素诱导的自噬过程中定位了这些Atgp-vYFP嵌合体。为了进一步说明该集合作为探索蛋白质在途径中的功能和相互作用的工具的效用，我们将这些ATG-vYFP嵌合体的子集定位在一个缺失支架蛋白Atg11p的菌株中。此外，我们验证了先前的结果，即在ATG11过表达和Atg1缺失时，在自噬前结构中鉴定出完整的膜蛋白Atg9p。总而言之，这种基于质粒的酵母基因-vYFP融合资源为探索萌芽酵母途径生物学的各种系统和大规模定位研究提供了初始工具箱。
Plasmid-based collections of fluorescent protein fusions are valuable and versatile resources, facilitating systematic studies of protein localization in multiple genetic backgrounds. At present, however, few such collections exist for the analysis of protein localization in any organism. To address this deficiency, we present here a plasmid-based set of resources for the analysis of protein localization in the budding yeast. Specifically, we constructed a suite of low-copy destination vectors for recombination-based cloning of yeast genes as fluorescent protein fusions. We cloned a set of 384 yeast genes encoding kinases, transcription factors and signaling proteins as "recombination-ready' cassettes; by Gateway cloning, these genes with native promoters can be easily introduced into the destination vectors described above, generating carboxy-terminal fusions to fluorescent proteins. Using these reagents, we constructed a subcollection of 276 genes encoding carboxy-terminal fusions to yellow fluorescent protein (vYFP). This collection encompasses 14 autophagy-related (ATG) genes, and we localized these Atgp-vYFP chimeras during rapamycin-induced autophagy. To illustrate further the utility of this collection as a tool in exploring the functions and interactions of proteins in a pathway, we localized a subset of these Atg-vYFP chimeras in a strain deleted for the scaffolding protein Atg11p. In addition, we validated previous results identifying the integral membrane protein Atg9p at the pre-autophagosomal structure upon overexpression of ATG11 and upon deletion of ATG1. Collectively, this plasmid-based resource of yeast gene-vYFP fusions provides an initial toolkit for a variety of systematic and large-scale localization studies exploring pathway biology in the budding yeast.