Functional analysis of the sumoylation pathway in Saccharomyces cerevisae

泛素和泛素样蛋白（例如SUMO-1）通过可逆的翻译后修饰调节细胞中的蛋白质功能。与泛素化不同，泛素化靶向蛋白质进行蛋白质体依赖性降解，类小泛素化被认为调节靶蛋白的细胞内位置、蛋白质-蛋白质相互作用和稳定性。然而，只有少数SUMO-1的底物蛋白是已知的，但预计许多SUMO-1携带靶标存在于酵母和后生动物中。因此，广泛的SUMO-1底物蛋白的鉴定有望深入了解真核细胞中SUMO化的调控方面。我们已经进行了蛋白质组范围内的分析sumoylated底物蛋白在酵母。采用一种有效的亲和纯化ProtA-Smt 3（Smt 3是酵母同源物的SUMO-1）从酵母裂解物结合质谱法，我们已经分离出100个潜在的Smt 3携带底物蛋白，并显示其中的代表性数量，他们确实携带SUMO-1的修改。值得注意的是，类小泛素化蛋白质的列表指出了所有主要细胞途径中的不同功能，代谢，抗氧化，蛋白水解，DNA复制和修复，染色质组织，转录激活，PolI，PolII和PolIII转录，5 '-前mRNA加帽，3'-前mRNA加工，蛋白酶体功能，染色质重塑，肌动蛋白和微管蛋白折叠，氨酰化，翻译，分泌，液泡功能和细胞组织。总之，我们的研究结果表明，类小泛素化是一个重要的机制，以调节和协调不同的细胞途径在真核生物。在我们未来的研究中，我们的目标是深入了解这种翻译后修饰对靶蛋白的功能意义，这些蛋白与基因表达有关。

Ubiquitin and ubiquitin-like proteins (e.g. SUMO-1) modulate protein function in the cell via reversible post-translational modification. Unlike ubiquitinylation, which targets proteins for proteosome-dependent degradation, sumoylation is thought to modulate the intracellular location, protein-protein interaction and stability of target proteins. However, only a few substrate proteins for SUMO-1 are known, but it is expected that many SUMO-1 carrying targets exist in yeast and metazoans. Thus, the identification of a broad range of SUMO-1 substrate proteins is expected to give insight into the regulatory aspects of sumoylation in a eukaryotic cell. We have performed a proteome-wide analysis of sumoylated substrate proteins in yeast. Employing an efficient affinity-purification of ProtA-Smt3 (Smt3 is the yeast homologue of SUMO-1) from yeast lysates in combination with mass spectrometry, we have isolated 100 potential Smt3-carrying substrate proteins and show for a representative number of them that they indeed carry the SUMO-1 modification. Notably, the list of sumoylated proteins points to diverse functions in all major cellular pathways, metabolism, anabolism, proteolysis, DNA replication and repair, chromatin organization, transcription activation, PolI, PolII and PolIII transcription, 5'-pre-mRNA capping, 3'-pre-mRNA processing, proteasome function, chromatin remodeling, actin and tubulin folding, aminoacylation, translation, secretion, vacuolar function and cellular organization. In overall, our findings suggest that sumoylation is an important mechanism to regulate and coordinate diverse cellular pathways in eukaryotes. In our future research, we aim to gain insight into the functional significance of this post-translational modification to target proteins, which are connected to gene expression.