Divergent modes of autophagy in the methylotrophic yeast Pichia pastoris.
Divergent modes of autophagy in the methylotrophic yeast Pichia pastoris.
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发表时间：
1995
影响因子：
4
通讯作者：
D. L. Tuttle;W. Dunn
中科院分区：
文献类型：
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作者：
D. L. Tuttle;W. Dunn
芽殖酵母巴斯德毕赤酵母通过合成高水平的胞质酶（例如甲酸脱氢酶）和过氧化物酶体酶（例如醇氧化酶）来响应甲醇培养基，这是同化这种碳源所必需的。细胞代谢的主要改变是在碳源向乙醇或葡萄糖转移时开始的。这些改变需要合成新的蛋白质和快速降解那些不再需要甲醇利用的酶。在这项研究中，我们已经测量了细胞溶质和过氧化物酶体的酶活性，并检查了形态上不同的过氧化物酶体的命运，以评估这种酵母在营养适应过程中的降解反应。利用生物化学、形态学和遗传学的方法，我们已经证明，在毕赤酵母中存在至少两条将过氧化物酶体隔离到液泡中进行降解的途径。乙醇诱导的途径是独立的蛋白质合成，并包括一个中间阶段，其中个别过氧化物酶体被隔离成自噬体的包装膜，然后融合的液泡。这个过程类似于macroautophagy。葡萄糖诱导的途径通过一个类似于微自噬的过程，通过液泡的指状突起引起过氧化物酶体簇的吞噬。与乙醇适应不同，葡萄糖也刺激甲酸脱氢酶的降解。过氧化物酶体保持在葡萄糖适应环己酰亚胺处理的正常细胞的液泡外，表明蛋白质合成是过氧化物酶体进入酵母液泡所必需的。两个互补的突变体（gsa 1和gsa 2），不能降解过氧化物酶体或甲酸脱氢酶在葡萄糖适应分离。突变的基因产物似乎在液泡内降解的一个或多个上游事件中起作用，因为乙醇诱导的过氧化物酶体降解在这些突变体中正常进行，并且在葡萄糖适应的gsa 2细胞的液泡外发现过氧化物酶体。缺乏液泡蛋白酶A和B的突变体不能降解乙醇氧化酶或甲酸脱氢酶在乙醇或葡萄糖适应。过氧化物酶体被发现积累在这些蛋白酶突变体的液泡在适应过程中。结合起来，结果表明，在巴斯德毕赤酵母中存在两个独立的途径，用于将过氧化物酶体隔离到液泡中，即降解位点。
The budding yeast Pichia pastoris responds to methanolic media by synthesizing high levels of cytosolic enzymes (e.g. formate dehydrogenase) and peroxisomal enzymes (e.g. alcohol oxidase), which are necessary to assimilate this carbon source. Major alterations in cellular metabolism are initiated upon a shift in carbon source to ethanol or glucose. These alterations require the synthesis of new proteins and the rapid degradation of those enzymes no longer needed for methanol utilization. In this study, we have measured cytosolic and peroxisomal enzyme activities and examined the fate of morphologically distinct peroxisomes to assess the degradative response of this yeast during nutrient adaptation. Utilizing biochemical, morphological and genetic approaches, we have shown that there exist in P. pastoris at least two pathways for the sequestration of peroxisomes into the vacuole for degradation. The ethanol-induced pathway is independent of protein synthesis and includes an intermediate stage in which individual peroxisomes are sequestered into autophagosomes by wrapping membranes, which then fuse with the vacuole. This process is analogous to macroautophagy. The glucose-induced pathway invokes the engulfment of clusters of peroxisomes by finger-like protrusions of the vacuole by a process analogous to microautophagy. Unlike ethanol adaptation, glucose stimulated the degradation of formate dehydrogenase as well. Peroxisomes remained outside the vacuoles of glucose-adapted cycloheximide-treated normal cells, suggesting that protein synthesis is required for peroxisome entry into the yeast vacuole. Two complementary mutants (gsa1 and gsa2) that are unable to degrade peroxisomes or formate dehydrogenase during glucose adaptation were isolated. The mutated gene products appear to function in one or more events upstream of degradation within the vacuole, since ethanol-induced peroxisome degradation proceeded normally in these mutants and peroxisomes were found outside the vacuoles of glucose-adapted gsa2 cells. Mutants lacking vacuolar proteinases A and B were unable to degrade alcohol oxidase or formate dehydrogenase during ethanol or glucose adaptation. Peroxisomes were found to accumulate within the vacuoles of these proteinase mutants during adaptation. Combined, the results suggest that there exist in Pichia pastoris two independent pathways for the sequestration of peroxisomes into the vacuole, the site of degradation.