Quantification of GTPase Cycling Rates of GTPases and GTPase:Effector Mixtures Using GTPase Glo Assays.

In different cellular activities such as signal transduction, cell division, and intracellular transportation, small guanosine triphosphatases (GTPases) take on a vital role. Their function involves hydrolysis of guanosine triphosphate (GTP) to guanosine diphosphate (GDP). In this article, we explain the application of a commercially available GTPase assay-the GTPase Glo assay by Promega-for investigation of GTPase-effector interactions. We provide experimental protocols together with an analysis model and software to obtain GTPase cycling rates of GTPases and GTPase:effector mixtures. GTPase cycling rates refer to the rates by which a GTPase completes an entire GTPase cycle. These rates enable quantification of the strength of GTPase effectors in a concentration-dependent fashion, as well as quantification of the combined effect of two effectors, independent of which GTPase cycle step they are affecting. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Conducting GTPase Glo assays Support Protocol 1: Analyzing GTPase assays to correlate luminescence with remaining GTP Support Protocol 2: Fitting GTPase assay data to obtain GTPase cycling rates.

Redundancy and the role of protein copy numbers in the cell polarization machinery of budding yeast.

How can a self-organized cellular function evolve, adapt to perturbations, and acquire new sub-functions? To make progress in answering these basic questions of evolutionary cell biology, we analyze, as a concrete example, the cell polarity machinery of Saccharomyces cerevisiae. This cellular module exhibits an intriguing resilience: it remains operational under genetic perturbations and recovers quickly and reproducibly from the deletion of one of its key components. Using a combination of modeling, conceptual theory, and experiments, we propose that multiple, redundant self-organization mechanisms coexist within the protein network underlying cell polarization and are responsible for the module's resilience and adaptability. Based on our mechanistic understanding of polarity establishment, we hypothesize that scaffold proteins, by introducing new connections in the existing network, can increase the redundancy of mechanisms and thus increase the evolvability of other network components. Moreover, our work gives a perspective on how a complex, redundant cellular module might have evolved from a more rudimental ancestral form.