In most Eukaryotes, ubiquitin either exists as free monoubiquitin or as a molecule that is covalently linked to other proteins. These two forms cycle between each other and due to the concerted antagonistic activity of ubiquitylating and deubiquitylating enzymes, an intracellular ubiquitin equilibrium is maintained that is essential for normal biological function. However, measuring the level and ratio of these forms of ubiquitin has been difficult and time consuming. In this paper, we have adapted a simple immunoblotting technique to monitor ubiquitin content and equilibrium dynamics in different developmental stages and tissues of Drosophila. Our data show that the level of total ubiquitin is distinct in different developmental stages, lowest at the larval-pupal transition and in three days old adult males, and highest in first instar larvae. Interestingly, the ratio of free mono-ubiquitin remains within 30–50% range of the total throughout larval development, but peaks to 70–80% at the larval-pupal and the pupal-adult transitions. It stays within the 70–80% range in adults. In developmentally and physiologically active tissues, the ratio of free ubiquitin is similarly high, most likely reflecting a high demand for ubiquitin availability. We also used this method to demonstrate the disruption of the finely tuned ubiquitin equilibrium by the abolition of proteasome function or the housekeeping deubiquitylase, Usp5. Our data support the notion that the ubiquitin equilibrium is regulated by tissue- and developmental stage-specific mechanisms.

在大多数真核生物中，泛素要么以游离的单泛素形式存在，要么以与其他蛋白质共价连接的分子形式存在。这两种形式彼此之间循环，并且由于泛素化和去泛素化酶的协同拮抗活性，维持了细胞内泛素平衡，这对于正常的生物学功能是必不可少的。然而，测量这些形式的遍在蛋白的水平和比例是困难且耗时的。在本文中，我们采用了一种简单的免疫印迹技术来监测果蝇不同发育阶段和组织中的遍在蛋白含量和平衡动力学。。我们的数据表明，在不同的发育阶段，总遍在蛋白的水平是不同的，在幼虫-pu过渡期和三日龄成年雄性中最低，而在第一龄幼虫中最高。有趣的是，在整个幼虫发育过程中，游离单泛素的比例保持在总量的30-50％范围内，但在幼虫-和and-成人过渡期达到峰值70-80％。在成年人中，它保持在70-80％的范围内。在具有发育和生理活性的组织中，游离泛素的比例同样很高，很可能反映了对泛素可用性的高需求。我们还使用这种方法来证明通过取消蛋白酶体功能或管家去泛素化酶Usp5破坏了微调的泛素平衡。