Collaborative Research: How Single Genes Provide Proteins to Multiple Cellular Compartments

合作研究：单基因如何向多个细胞区室提供蛋白质

• 批准号: 0115409
• 负责人: Anita Hopper
• 金额: $ 12.67万
• 依托单位: Pennsylvania State Univ University Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-01 至 2004-02-29
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0115409&HistoricalAwards=false
• 关键词: Collaborative; Research; How; Single; Genes

This award supports a collaborative research program from Dr. Anita K. Hopper, Penn. State Univ. Coll. Medicine and Dr. Nancy C. Martin, Univ. Louisville that will examine how single genes provide proteins to more than one compartment within cells. The discovery that this occurs emerged from observations that mutations affecting cytosolic tRNA modification also affect mitochondrial tRNA. The enzymes making these modifications were called sorting isozymes because they came from a single gene and carried out the same function in multiple cellular compartments. Previous results from these investigators showed that by alternative use of in-frame translation initiation codons the yeast genes TRM1, MOD5 and CCA1 each encode multiple isozymes. For each, the isozyme initiating at the first initiator codon is mitochondrial. However, for Trm1p the amino-terminal extension is not essential for mitochondrial import and for Mod5p the long form is also found in the cytosol. The shorter forms of the proteins can be located in nuclei and/or the cytosol. Earlier results from this collaborative project showed: (1) organelles can share information with the rest of the cell; (2) isozymes can be located in more than one compartment; (3) sorting information resides in additional sequences missing from the eubacterial and archaeal counterparts; (4) compartments may have activity provided by more than one isozyme; (5) mitochondrial targeting information located at amino termini prohibits nuclear import. Despite the advances, important questions remain because the mechanisms that underlie #4 and #5 are still not understood. Aim I is designed to understand how Trm1p can possess both mitochondrial and nuclear targeting information and yet not locate to the nucleus. Possible mechanisms by which commitment to mitochondrial import occurs will be tested using a novel system developed by the investigators that assesses where in a cell a protein destined to mitochondria folds. Experiments will also be performed to learn whether mRNA sorting, cytosolic retention, and/or nuclear export function to prohibit nuclear accumulation of Trm1p-I. Sorting isozymes are not only distributed to different organelles; they have distinct suborganellar locations. For example, the investigators showed that two sorting isozymes have distinct subnuclear locations; Trm1p, is located at the inner nuclear membrane (INM) and Mod5p is located in the nucleoplasm and the nucleolus. Goals of Aim II are to confirm and characterize motif(s) specifying INM location and to identify gene products that tether Trm1p to the INM. Yeast mutants that fail to appropriately locate Trm1p to the INM will be characterized. The studies in Aim II will not only address how sorting isozymes locate to the correct suborganellar locations, but, in addition, will provide information concerning nuclear architecture and nuclear biogenesis - an important area of cell biology not understood for any eukaryote.

该奖项支持Anita K博士的合作研究计划。霍普，佩恩。美国州立大学医学院和南希·C.马丁，路易斯维尔大学，将研究单个基因如何提供蛋白质到细胞内的一个以上的隔室。这一发现来自于观察到影响胞质tRNA修饰的突变也影响线粒体tRNA。 进行这些修饰的酶被称为分选同工酶，因为它们来自单个基因，在多个细胞区室中执行相同的功能。这些研究人员先前的结果表明，通过交替使用框内翻译起始密码子，酵母基因TRM 1、MOD 5和CCA 1各自编码多种同工酶。对于每一种，在第一个起始密码子处起始的同工酶是线粒体的。然而，对于Trm 1 p的氨基末端的延伸是不是必不可少的线粒体输入和Mod 5 p的长形式也被发现在胞质溶胶。较短形式的蛋白质可以位于细胞核和/或细胞质中。这个合作项目的早期结果表明：（1）细胞器可以与细胞的其他部分共享信息;（2）同工酶可以位于一个以上的区室中;（3）分选信息存在于真细菌和古细菌对应物中缺失的额外序列中;（4）区室可以由一种以上的同工酶提供活性;（5）位于氨基末端的线粒体靶向信息禁止核输入。尽管取得了进展，但重要的问题仍然存在，因为#4和#5的基础机制仍然不清楚。 目的研究Trm 1 p是如何同时具有线粒体和核靶向信息而不定位于细胞核的。将使用研究人员开发的一种新系统来测试线粒体输入发生的可能机制，该系统评估细胞中蛋白质在线粒体折叠的位置。还将进行实验以了解mRNA分选、胞质保留和/或核输出功能是否抑制Trm 1 p-I的核蓄积。分选同工酶不仅分布在不同的细胞器中，而且具有不同的亚细胞器位置。例如，研究人员发现两种分选同工酶具有不同的亚核位置; Trm 1 p位于内核膜（INM），Mod 5 p位于核质和核仁中。 目的II的目标是确认和表征指定INM位置的基序，并鉴定将Trm 1 p连接到INM的基因产物。将表征不能将Trm 1 p适当定位于INM的酵母突变体。目标II中的研究不仅将解决如何分选同工酶定位到正确的亚细胞器位置，而且还将提供有关核结构和核生物发生的信息-这是任何真核生物都不了解的细胞生物学的重要领域。