High-Resolution Membrane Capacitance Measurements for Studying Endocytosis and Exocytosis in Yeast

Abstract

With patch-clamp recording, we detect in yeast protoplasts individual exo- and endocytotic events as discrete steps in membrane capacitance. The high-resolution data show that exo- and endocytotic vesicles undergo, similar to other eukaryotes, permanent and transient fusion/fission with a bias for transient events. The electrical data are a good representation of the membrane dynamics of a growing yeast cell. Fusion of exocytotic vesicles with the plasma membrane gives rise to an increase in membrane surface area, whereas the surface area is decreased when vesicles are internalized during endocytosis. Changes in membrane surface area, resulting from fusion and fission of membrane vesicles, can be followed by monitoring the corresponding proportional changes in membrane capacitance. Using the cell-attached configuration of the patch-clamp techniques we were able to resolve the elementary processes of endo- and exocytosis in yeast protoplasts at high temporal and spatial resolution. Spontaneous capacitance changes were predominantly in the range of 0.2-1 fF which translates to vesicle diameters of 90-200nm. The size distribution revealed that endocytotic vesicles with a median at about 132nm were smaller than exocytotic vesicles with a median at 155nm. In energized and metabolizing protoplasts, endo- and exocytotic events occurred at frequencies of 1.6 and 2.7 events per minute, respectively. Even though these numbers appear very low, they are in good agreement with the observed growth rate of yeast cells and protoplasts.