Establishing Regulation of a Dynamic Process by Ypt/Rab GTPases : A Case for Cisternal Progression.

The prevailing model for transport within the Golgi is cisternal maturation. This process can be viewed as switching of cisternal markers using live-cell microscopy in yeast cells since the Golgi cisternae are separated, as opposed to the stacked Golgi seen in other organisms. It is also possible to determine which trafficking machinery components are required for this process by studying mutants depleted for these components. However, determining how cisternal maturation is regulated has been more challenging. The key for demonstrating regulation is not solely to stop the maturation when depleting a vesicular trafficking component, but also to illustrate a change in the speed. The obvious candidates for such regulation are the Ypt/Rab GTPases because of their established mode of action as regulators. Since the precise localization of the Golgi Ypts, Ypt1 and Ypt31, to specific Golgi cisternae has been controversial, we started by carefully colocalizing these Ypts with the Golgi cisternal markers using two independent approaches: immunofluorescence and live-cell microscopy. Next, the opposite effects of depletion versus constitutively activating Ypt mutations on Golgi morphology were determined. Finally, the ability of constitutively activating Ypt mutations to accelerate a specific cisternal-maturation step was established by live-cell time-lapse microscopy. Using these approaches, we defined three dynamic Golgi cisternae, early, intermediate, and late, separated two independent steps of cisternal maturation and showed their regulation by two different Ypts. Here, we discuss the major principles and precautions needed for each phase of this research, the main point being definition of a new criterion for regulation of a dynamic process versus requirement of a machinery structural component: acceleration of the dynamics.