Sakai Y, Koller A, Rangell L K, Keller G A, Subramani S
Department of Biology, University of California, San Diego, La Jolla, California 92093-0322, USA.
J Cell Biol. 1998 May 4;141(3):625-36. doi: 10.1083/jcb.141.3.625.
We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl ) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor-product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.
我们使用染料N-(3-三乙铵丙基)-4-(对二乙氨基苯基己三烯基)吡啶二溴化物（FM4-64）和一种融合蛋白（由连接到过氧化物酶体靶向信号Ser-Lys-Leu [SKL]的绿色荧光蛋白组成），分别对活体巴斯德毕赤酵母细胞中的液泡膜和过氧化物酶体基质进行标记，然后通过荧光显微镜观察过氧化物酶体通过微自噬和巨自噬在液泡降解过程中的形态和动力学中间体。通过电子显微镜也鉴定出了与这些中间体相对应的结构。这些中间体出现和消失的动力学与中间体之间的前体-产物关系一致，这构成了微自噬模型的基础。影响微自噬不同步骤的抑制剂并未损害过氧化物酶体通过巨自噬传递到液泡，尽管液泡蛋白酶的抑制通过两种自噬途径影响了绿色荧光蛋白（S65T突变体版本[GFP]）-SKL在液泡中的最终降解。分离并鉴定了在过氧化物酶体微自噬方面有缺陷的巴斯德毕赤酵母突变体（pag突变体）中是否存在这些中间体。这些突变体包括6个互补组，支持微自噬模型。我们的研究表明，过氧化物酶体的微自噬降解通过特定的中间体进行，其产生和/或加工受PAG基因产物控制，并为人们了解甚少的过氧化物酶体稳态现象提供了线索。
