van Zutphen Tim, van der Klei Ida J, Kiel Jan A K W
Molecular Cell Biology, University of Groningen, The Netherlands.
Methods Enzymol. 2008;451:197-215. doi: 10.1016/S0076-6879(08)03214-X.
In the yeast Hansenula polymorpha the development and turnover of peroxisomes is readily achieved by manipulation of the cultivation conditions. The organelles massively develop when the cells are incubated in the presence of methanol as the sole source of carbon and energy. However, they are rapidly and selectively degraded when methanol-grown cells are placed at conditions of repression of methanol metabolism (e.g. in glucose or ethanol excess conditions) by a process termed macropexophagy. Degradation of peroxisomes is also observed when the cells are placed at nitrogen-depletion conditions (microautophagy). This contribution details the methodologies that are currently in use investigating macropexophagy and microautophagy in H. polymorpha. Emphasis is placed on various structural (fluorescence microscopy, electron microscopy) and biochemical (specific enzyme activity measurements, Western blotting) approaches.
在多形汉逊酵母中,通过控制培养条件可以很容易地实现过氧化物酶体的发育和更新。当细胞在甲醇作为唯一碳源和能源的存在下孵育时,这些细胞器会大量发育。然而,当甲醇培养的细胞置于甲醇代谢受抑制的条件下(例如在葡萄糖或乙醇过量的条件下)时,它们会通过一种称为巨自噬的过程迅速且选择性地降解。当细胞置于氮耗尽条件下时,也会观察到过氧化物酶体的降解(微自噬)。本论文详细介绍了目前用于研究多形汉逊酵母中巨自噬和微自噬的方法。重点介绍了各种结构(荧光显微镜、电子显微镜)和生化(特定酶活性测量、蛋白质免疫印迹)方法。