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

该奖项支持宾夕法尼亚大学Anita K. Hopper博士的合作研究项目。州立大学学院医学和路易斯维尔大学的Nancy C. Martin博士将研究单个基因如何为细胞内的多个隔间提供蛋白质。这一发现来自于对影响胞质tRNA修饰的突变也影响线粒体tRNA的观察。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，并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，通过使用框架内翻译起始密码子，酵母基因TRM1、MOD5和CCA1各自编码多个同工酶。对于每一种，在第一个启动密码子处启动的同工酶是线粒体的。然而，对于Trm1p来说，氨基末端的延伸对于线粒体的输入并不是必需的，而对于Mod5p来说，长形式也存在于细胞质中。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)同工酶可位于多个隔室；(3)分类信息存在于真细菌和古细菌对应序列缺失的附加序列中；(4)隔室可能具有由一种以上同工酶提供的活性；(5)位于氨基末端的线粒体靶向信息阻止核输入。尽管取得了进展，但重要的问题仍然存在，因为第4条和第5条背后的机制仍然不清楚。Aim I旨在了解Trm1p如何同时拥有线粒体和核靶向信息而不定位到细胞核。研究人员将使用一种新系统来测试线粒体输入发生的可能机制，该系统可以评估细胞中注定要进入线粒体的蛋白质的折叠位置。实验还将进行，以了解mRNA分选，细胞质保留，和/或核输出功能是否阻止trmp1 - i的核积累。分选同工酶不仅分布在不同的细胞器上；它们有不同的细胞器亚位置。例如，研究人员发现，两种分选同工酶具有不同的亚核位置；Trm1p位于内核膜（INM）， Mod5p位于核质和核仁。Aim II的目标是确认和表征指定INM位置的基序，并鉴定将Trm1p连接到INM的基因产物。酵母突变体不能正确定位Trm1p到INM将被表征。Aim II的研究不仅将解决分选同工酶如何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。

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.