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

该奖项支持宾夕法尼亚州安妮塔·K·霍珀博士的一项合作研究计划。州立大学。科尔。医学和南希·C·马丁博士，大学他将研究单个基因如何将蛋白质提供给细胞内的多个隔室。发生这种情况的发现源于观察到影响胞质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类同工酶，因为它们来自单一基因，在多个细胞隔间执行相同的功能。这些研究人员之前的研究结果表明，通过替换使用框内翻译起始密码子，酵母基因TRM1、MOD5和CCA1各自编码多个同工酶。对于每一种，在第一个启动子密码子上启动的同工酶是线粒体。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不可少的，而对于mod5p来说，细胞质中也发现了长的形式。较短形式的蛋白质可以位于细胞核和/或胞浆中。这一合作项目的早期结果表明：(1)细胞器可以与细胞的其余部分共享信息；(2)同工酶可以位于多个隔室；(3)分类信息存在于真细菌和古生菌对应的缺失的额外序列中；(4)隔室可能具有由多个同工酶提供的活性；(5)位于氨基末端的线粒体靶向信息禁止核输入。尽管取得了这些进展，但重要的问题仍然存在，因为#4和#5背后的机制仍然不清楚。目的I旨在了解Trm1p如何同时拥有线粒体和核靶向信息，而又不定位于细胞核。将使用研究人员开发的一种新系统来测试承诺线粒体输入的可能机制，该系统评估指定用于线粒体的蛋白质在细胞中的哪里折叠。还将进行实验，以了解mRNA分选、胞质滞留和/或核输出功能是否能够阻止Trm1p-I的核积累。分选同工酶不仅分布在不同的细胞器中，它们还具有不同的细胞器下位置。例如，研究人员发现，两种分类同工酶具有不同的亚核位置；Trm1p位于内核膜(INM)，而mod5p位于核浆和核仁中。AIM II的目标是确认和鉴定指定INM位置的基序(S)，并鉴定将Trm1p拴在INM上的基因产物。未能将Trm1p适当定位到INM的酵母突变株将被鉴定。AIM II上的研究不仅将解决如何将分选同工酶定位到正确的细胞器下部位置，而且还将提供关于核结构和核生物发生的信息--这是一个对任何真核生物都不了解的重要细胞生物学领域。

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.