Yuan W, Tuttle D L, Shi Y J, Ralph G S, Dunn W A
Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville 32610, USA.
J Cell Sci. 1997 Aug;110 ( Pt 16):1935-45. doi: 10.1242/jcs.110.16.1935.
We have characterized biochemically, morphologically, and genetically two distinct pathways for the selective degradation of peroxisomes in Pichia pastoris. These pathways are independently regulated and analogous to microautophagy and macroautophagy that have been defined in mammalian cells. When P. pastoris is grown in methanol, cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized. During adaptation from methanol to glucose, these enzymes are rapidly and selectively degraded within the yeast vacuole by microautophagy. We have isolated gsa mutants that are defective in glucose-induced selective autophagy of peroxisomes. In this study, we have shown that gsa1 is unable to sequester peroxisomes into the yeast vacuole. In addition, we provide evidence that the glucose-induced selective autophagy 1 (GSA1) protein is the alpha subunit of the phosphofructokinase enzyme complex encoded by PFK1. First, we can rescue the gsa1 mutant by transformation with a vector containing PFK1. Second, cellular levels of both PFK1 mRNA and phosphofructokinase activity are dramatically reduced in gsa1 when compared to the parental GS115. Third, a PFK1 knockout (delta pfk1) is unable to degrade alcohol oxidase during glucose adaptation. As observed in gsa1, the peroxisomes in delta pfk1 remain outside the vacuole during adaptation. Our data are consistent with the concept that PFK1 protein is required for an event upstream of vacuole degradation (i.e. signaling, selection, or sequestration). However, the degradation of peroxisomes does not require a catalytically active phosphofructokinase. The inability of delta pfk1 cells to degrade alcohol oxidase can be rescued by transformation with either normal PFK1 or mutant pfk1 whose catalytic site had been inactivated by a single amino acid mutation. We propose that PFK1 protein directly modulates glucose-induced microautophagy independent of its ability to metabolize glucose intermediates.
我们已从生物化学、形态学和遗传学角度对毕赤酵母中过氧化物酶体选择性降解的两条不同途径进行了表征。这些途径受独立调控，类似于在哺乳动物细胞中定义的微自噬和巨自噬。当毕赤酵母在甲醇中生长时，会合成甲醇同化所需的胞质和过氧化物酶体酶。在从甲醇适应葡萄糖的过程中，这些酶会通过微自噬在酵母液泡中迅速且选择性地降解。我们分离出了在葡萄糖诱导的过氧化物酶体选择性自噬中存在缺陷的gsa突变体。在本研究中，我们表明gsa1无法将过氧化物酶体隔离到酵母液泡中。此外，我们提供证据表明葡萄糖诱导的选择性自噬1（GSA1）蛋白是由PFK1编码的磷酸果糖激酶酶复合物的α亚基。首先，我们可以通过用含有PFK1的载体转化来拯救gsa1突变体。其次，与亲本GS115相比，gsa1中PFK1 mRNA和磷酸果糖激酶活性的细胞水平均显著降低。第三，PFK1基因敲除（delta pfk1）在葡萄糖适应过程中无法降解醇氧化酶。如在gsa1中观察到的那样，delta pfk1中的过氧化物酶体在适应过程中仍留在液泡外。我们的数据与PFK1蛋白是液泡降解上游事件（即信号传导、选择或隔离）所必需的这一概念一致。然而，过氧化物酶体的降解并不需要具有催化活性的磷酸果糖激酶。用正常的PFK1或其催化位点已因单个氨基酸突变而失活的突变体pfk1转化，可以拯救delta pfk1细胞降解醇氧化酶的能力。我们提出PFK1蛋白直接调节葡萄糖诱导的微自噬，而与其代谢葡萄糖中间体的能力无关。
