Tuttle D L, Dunn W A
University of Florida College of Medicine, Department of Anatomy and Cell Biology, Gainesville 32610-0235, USA.
J Cell Sci. 1995 Jan;108 ( Pt 1):25-35. doi: 10.1242/jcs.108.1.25.
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.
出芽酵母巴斯德毕赤酵母通过合成高水平的胞质酶（如甲酸脱氢酶）和过氧化物酶体酶（如乙醇氧化酶）来响应甲醇培养基，这些酶是同化这种碳源所必需的。当碳源转变为乙醇或葡萄糖时，细胞代谢会发生重大改变。这些改变需要合成新的蛋白质，并快速降解那些不再用于甲醇利用的酶。在本研究中，我们测量了胞质和过氧化物酶体酶的活性，并研究了形态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。利用生化、形态学和遗传学方法，我们发现巴斯德毕赤酵母中至少存在两条将过氧化物酶体隔离到液泡中进行降解的途径。乙醇诱导的途径不依赖蛋白质合成，包括一个中间阶段，在此阶段单个过氧化物酶体通过包裹膜被隔离到自噬体中，然后自噬体与液泡融合。这个过程类似于巨自噬。葡萄糖诱导的途径通过液泡的指状突起以类似于微自噬的过程吞噬过氧化物酶体簇。与乙醇适应不同，葡萄糖也刺激了甲酸脱氢酶的降解。过氧化物酶体留在经环己酰亚胺处理的葡萄糖适应正常细胞的液泡外，这表明过氧化物酶体进入酵母液泡需要蛋白质合成。分离出了两个互补突变体（gsa1和gsa2），它们在葡萄糖适应过程中无法降解过氧化物酶体或甲酸脱氢酶。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gsa2细胞的液泡外发现了过氧化物酶体。缺乏液泡蛋白酶A和B的突变体在乙醇或葡萄糖适应过程中无法降解乙醇氧化酶或甲酸脱氢酶。在适应过程中，过氧化物酶体在这些蛋白酶突变体的液泡内积累。综合来看，结果表明巴斯德毕赤酵母中存在两条独立的将过氧化物酶体隔离到液泡（降解位点）中的途径。
