Structural Disorder Provides Increased Adaptability for Vesicle Trafficking Pathways

Vesicle trafficking systems play essential roles in the communication between the organelles of eukaryotic cells and also between cells and their environment. Endocytosis and the late secretory route are mediated by clathrin-coated vesicles, while the COat Protein I and II (COPI and COPII) routes stand for the bidirectional traffic between the ER and the Golgi apparatus. Despite similar fundamental organizations, the molecular machinery, functions, and evolutionary characteristics of the three systems are very different. In this work, we compiled the basic functional protein groups of the three main routes for human and yeast and analyzed them from the structural disorder perspective. We found similar overall disorder content in yeast and human proteins, confirming the well-conserved nature of these systems. Most functional groups contain highly disordered proteins, supporting the general importance of structural disorder in these routes, although some of them seem to heavily rely on disorder, while others do not. Interestingly, the clathrin system is significantly more disordered (,23%) than the other two, COPI (,9%) and COPII (,8%). We show that this structural phenomenon enhances the inherent plasticity and increased evolutionary adaptability of the clathrin system, which distinguishes it from the other two routes. Since multi-functionality (moonlighting) is indicative of both plasticity and adaptability, we studied its prevalence in vesicle trafficking proteins and correlated it with structural disorder. Clathrin adaptors have the highest capability for moonlighting while also comprising the most highly disordered members. The ability to acquire tissue specific functions was also used to approach adaptability: clathrin route genes have the most tissue specific exons encoding for protein segments enriched in structural disorder and interaction sites. Overall, our results confirm the general importance of structural disorder in vesicle trafficking and suggest major roles for this structural property in shaping the differences of evolutionary adaptability in the three routes

Optimization of a blueprint for in vitro glycolysis by metabolic real-time analysis

Recruiting complex metabolic reaction networks for chemical synthesis has attracted considerable attention but frequently requires optimization of network composition and dynamics to reach sufficient productivity. As a design framework to predict optimal levels for all enzymes in the network is currently not available, state-of-the-art pathway optimization relies on high-throughput phenotype screening. We present here the development and application of a new in vitro real-time analysis method for the comprehensive investigation and rational programming of enzyme networks for synthetic tasks. We used this first to rationally and rapidly derive an optimal blueprint for the production of the fine chemical building block dihydroxyacetone phosphate (DHAP) via Escherichia coli’s highly evolved glycolysis. Second, the method guided the three-step genetic implementation of the blueprint, yielding a synthetic operon with the predicted 2.5-fold–increased glycolytic flux toward DHAP. The new analytical setup drastically accelerates rational optimization of synthetic multienzyme networks.