Molecular mechanisms of endo- and exocytosis in the baker's yeast

胞内和胞吐作用在真核生物的许多细胞过程中是必不可少的，例如极性生长、细胞分裂、运动或信号传导。通过胞吐囊泡的融合，细胞将分泌物、膜驻留蛋白以及膜递送至质膜;这些物质的逆向运输由胞吞囊泡的裂变提供。由于细胞膜的电性质由选择性电导和电容组成，后者与膜表面积成比例，因此可以通过记录膜电容直接监测囊泡融合和分裂引起的表面积变化。通过这种方式，可以在活细胞中真实的时间内记录单个胞外和胞吞囊泡的大小及其融合和分裂的动力学。在表明这种记录技术在酵母原生质体中也是可行的之后，我们将在该提议中联合收割机将酵母中关于胞外/内吞的详细遗传和生物化学知识与高分辨率电容记录相结合。利用丰富的酵母突变体库，在胞外和胞吞机制中的不同蛋白质中存在缺陷，并利用成熟的膜运输信号（如交配信息素），将有可能将融合和裂变的复杂机制分解为明确的分子步骤。由于分子机制的保守性，这项工作的结果将对理解哺乳动物细胞中的这些过程产生强烈的影响。

Endo- and exocytosis are imperative in numerous cellular processes in eukaryotes, such as polar growth, cell division, motility or signaling. By fusion of exocytotic vesicles cells deliver secretory cargo, membrane-resident proteins and also membrane to the plasma membrane; the retrogarde transport of these materials is provided by fisson of endocytotic vesicles. Since the electrical properties of a cell membrane are comprised of a selective conductance and a capacitance, the latter being proportional to the membrane surface area, changes in the surface area from fusion and fission of vesicles can be directly monitored by recording of the membrane capacitance. In this way the size of individual exo- and endocytotic vesicles and their kinetics of fusion and fission can be recorded in real time in living cells. After showing that this recording technique is also feasible in yeast protoplasts we will in this proposal combine the detailed genetic and biochemical knowledge on exo-/endocytosis in yeast with high resolution capacitance recordings. The use of the rich library of yeast mutants with defects in distinct proteins in the exo- and endocytotic machinery and the use of well established signals for membrane trafficking like the mating pheromones will make it possible to dissect the complex mechanisms of fusion and fission into well defined molecular steps. Because of the conservation of the molecular machinery the results of this work will have strong implications for the understanding of these processes in mammalian cells.