Abstract
Exo- and endocytosis are crucial in numerous physiological processes in eukaryotic cells, such as polar growth, cell division, motility or signaling. Endo- as well as exocytotic events result in small changes of the cell surface area, which can be resolved in electrophysiological recordings in the form of discrete steps in membrane capacitance. By applying high resolution recordings with yeast protoplasts, we were able to resolve discrete changes in membrane capacitance mainly in the range 0.2-1.2fF, corresponding to fusion and fission of single vesicles of 80-250nm. Analysis of exo- and endocytosis in the yeast sec6-4 mutant, which is characterized by a temperature dependent block of exocytosis, revealed low fission/fusion activity at room temperature. Incubation at the restrictive temperature of 37°C for 3h and shifting to the permissive temperature of 25°C resulted in a transient increase in endo-/exocytotic activity of up to 10-fold. This study demonstrates that patch clamp capacitance measurements in yeast provide a novel in vivo tool to examine the molecular mechanisms of endo-/exocytosis in this model organism. The recordings gain information in real time on the geometry of endo-/exocytotic vesicles, on the kinetics of the events and their dependence on signaling cascades and key proteins.
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