Yuan W, Stromhaug P E, Dunn W A
Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, Florida 32610, USA.
Mol Biol Cell. 1999 May;10(5):1353-66. doi: 10.1091/mbc.10.5.1353.
Cytosolic and peroxisomal enzymes necessary for methanol assimilation are synthesized when Pichia pastoris is grown in methanol. Upon adaptation from methanol to a glucose environment, these enzymes are rapidly and selectively sequestered and degraded within the yeast vacuole. Sequestration begins when the vacuole changes shape and surrounds the peroxisomes. The opposing membranes then fuse, engulfing the peroxisome. In this study, we have characterized a mutant cell line (glucose-induced selective autophagy), gsa7, which is defective in glucose-induced selective autophagy of peroxisomes, and have identified the GSA7 gene. Upon glucose adaptation, gsa7 cells were unable to degrade peroxisomal alcohol oxidase. We observed that the peroxisomes were surrounded by the vacuole, but complete uptake into the vacuole did not occur. Therefore, we propose that GSA7 is not required for initiation of autophagy but is required for bringing the opposing vacuolar membranes together for homotypic fusion, thereby completing peroxisome sequestration. By sequencing the genomic DNA fragment that complemented the gsa7 phenotype, we have found that GSA7 encodes a protein of 71 kDa (Gsa7p) with limited sequence homology to a family of ubiquitin-activating enzymes, E1. The knockout mutant gsa7Delta had an identical phenotype to gsa7, and both mutants were rescued by an epitope-tagged Gsa7p (Gsa7-hemagglutinin [HA]). In addition, a GSA7 homolog, APG7, a protein required for autophagy in Saccharomyces cerevisiae, was capable of rescuing gsa7. We have sequenced the human homolog of GSA7 and have shown many regions of identity between the yeast and human proteins. Two of these regions align to the putative ATP-binding domain and catalytic site of the family of ubiquitin activating enzymes, E1 (UBA1, UBA2, and UBA3). When either of these sites was mutated, the resulting mutants [Gsa7(DeltaATP)-HA and Gsa7(C518S)-HA] were unable to rescue gsa7 cells. We provide evidence to suggest that Gsa7-HA formed a thio-ester linkage with a 25-30 kDa protein. This conjugate was not observed in cells expressing Gsa7(DeltaATP)-HA or in cells expressing Gsa7(C518S)-HA. Our results suggest that this unique E1-like enzyme is required for homotypic membrane fusion, a late event in the sequestration of peroxisomes by the vacuole.
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。从甲醇环境适应到葡萄糖环境后，这些酶会迅速且有选择性地被隔离并在酵母液泡内降解。当液泡改变形状并包围过氧化物酶体时，隔离开始。然后相对的膜融合，吞噬过氧化物酶体。在本研究中，我们鉴定了一种突变细胞系（葡萄糖诱导的选择性自噬）gsa7，它在葡萄糖诱导的过氧化物酶体选择性自噬方面存在缺陷，并鉴定了GSA7基因。在适应葡萄糖后，gsa7细胞无法降解过氧化物酶体乙醇氧化酶。我们观察到过氧化物酶体被液泡包围，但没有完全被液泡摄取。因此，我们提出GSA7不是自噬起始所必需的，但对于使相对的液泡膜聚集以进行同型融合从而完成过氧化物酶体隔离是必需的。通过对补充gsa7表型的基因组DNA片段进行测序，我们发现GSA7编码一种71 kDa的蛋白质（Gsa7p），与泛素激活酶家族E1的序列同源性有限。敲除突变体gsa7Delta具有与gsa7相同的表型，并且两种突变体都可被表位标记的Gsa7p（Gsa7-血凝素[HA]）拯救。此外，GSA7的同源物APG7，酿酒酵母中自噬所需的一种蛋白质，能够拯救gsa7。我们对GSA7的人类同源物进行了测序，并显示了酵母和人类蛋白质之间的许多相同区域。其中两个区域与泛素激活酶家族E1（UBA1、UBA2和UBA3）的推定ATP结合域和催化位点对齐。当这些位点中的任何一个发生突变时，产生的突变体[Gsa7(DeltaATP)-HA和Gsa7(C518S)-HA]无法拯救gsa7细胞。我们提供的证据表明Gsa7-HA与一种25 - 30 kDa的蛋白质形成了硫酯键。在表达Gsa7(DeltaATP)-HA的细胞或表达Gsa7(C518S)-HA的细胞中未观察到这种共轭物。我们的结果表明，这种独特的类E1酶是同型膜融合所必需的，这是液泡隔离过氧化物酶体过程中的一个晚期事件。
