Oku Masahide, Warnecke Dirk, Noda Takeshi, Müller Frank, Heinz Ernst, Mukaiyama Hiroyuki, Kato Nobuo, Sakai Yasuyoshi
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwake, Sakyo-ku, Kyoto 606-8502, Germany.
EMBO J. 2003 Jul 1;22(13):3231-41. doi: 10.1093/emboj/cdg331.
Fungal sterol glucosyltransferases, which synthesize sterol glucoside (SG), contain a GRAM domain as well as a pleckstrin homology and a catalytic domain. The GRAM domain is suggested to play a role in membrane traffic and pathogenesis, but its significance in any biological processes has never been experimentally demonstrated. We describe herein that sterol glucosyltransferase (Ugt51/Paz4) is essential for pexophagy (peroxisome degradation), but not for macroautophagy in the methylotrophic yeast Pichia pastoris. By expressing truncated forms of this protein, we determined the individual contributions of each of these domains to pexophagy. During micropexophagy, the glucosyltransferase was associated with a recently identified membrane structure: the micropexophagic apparatus. A single amino acid substitution within the GRAM domain abolished this association as well as micropexophagy. This result shows that GRAM is essential for proper protein association with its target membrane. In contrast, deletion of the catalytic domain did not impair protein localization, but abolished pexophagy, suggesting that SG synthesis is required for this process.
合成甾醇葡萄糖苷（SG）的真菌甾醇葡萄糖基转移酶含有一个GRAM结构域以及一个普列克底物蛋白同源结构域和一个催化结构域。GRAM结构域被认为在膜运输和致病过程中发挥作用，但其在任何生物学过程中的重要性从未得到实验证明。我们在此描述，甾醇葡萄糖基转移酶（Ugt51/Paz4）对甲基营养型酵母毕赤酵母中的过氧化物酶体自噬（过氧化物酶体降解）至关重要，但对巨自噬并非必需。通过表达该蛋白的截短形式，我们确定了这些结构域各自对过氧化物酶体自噬的贡献。在微过氧化物酶体自噬过程中，葡萄糖基转移酶与最近发现的一种膜结构：微过氧化物酶体自噬装置相关联。GRAM结构域内的单个氨基酸取代消除了这种关联以及微过氧化物酶体自噬。这一结果表明，GRAM对于蛋白质与其靶膜的正确结合至关重要。相比之下，催化结构域的缺失并不影响蛋白质定位，但消除了过氧化物酶体自噬，这表明该过程需要SG的合成。
