Santt Olivier, Pfirrmann Thorsten, Braun Bernhard, Juretschke Jeannette, Kimmig Philipp, Scheel Hartmut, Hofmann Kay, Thumm Michael, Wolf Dieter H
Institut für Biochemie, Universität Stuttgart, 70569 Stuttgart, Germany.
Mol Biol Cell. 2008 Aug;19(8):3323-33. doi: 10.1091/mbc.e08-03-0328. Epub 2008 May 28.
Glucose-dependent regulation of carbon metabolism is a subject of intensive studies. We have previously shown that the switch from gluconeogenesis to glycolysis is associated with ubiquitin-proteasome linked elimination of the key enzyme fructose-1,6-bisphosphatase. Seven glucose induced degradation deficient (Gid)-proteins found previously in a genomic screen were shown to form a complex that binds FBPase. One of the subunits, Gid2/Rmd5, contains a degenerated RING finger domain. In an in vitro assay, heterologous expression of GST-Gid2 leads to polyubiquitination of proteins. In addition, we show that a mutation in the degenerated RING domain of Gid2/Rmd5 abolishes fructose-1,6-bisphosphatase polyubiquitination and elimination in vivo. Six Gid proteins are present in gluconeogenic cells. A seventh protein, Gid4/Vid24, occurs upon glucose addition to gluconeogenic cells and is afterwards eliminated. Forcing abnormal expression of Gid4/Vid24 in gluconeogenic cells leads to fructose-1,6-bisphosphatase degradation. This suggests that Gid4/Vid24 initiates fructose-1,6-bisphosphatase polyubiquitination by the Gid complex and its subsequent elimination by the proteasome. We also show that an additional gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, is subject to Gid complex-dependent degradation. Our study uncovers a new type of ubiquitin ligase complex composed of novel subunits involved in carbohydrate metabolism and identifies Gid4/Vid24 as a major regulator of this E3.
葡萄糖依赖性碳代谢调节是深入研究的课题。我们之前已经表明，从糖异生向糖酵解的转变与泛素-蛋白酶体介导的关键酶果糖-1,6-二磷酸酶的消除有关。先前在基因组筛选中发现的七种葡萄糖诱导降解缺陷（Gid）蛋白形成了一个与果糖-1,6-二磷酸酶结合的复合物。其中一个亚基Gid2/Rmd5含有一个退化的RING指结构域。在体外试验中，GST-Gid2的异源表达导致蛋白质的多聚泛素化。此外，我们表明Gid2/Rmd5退化RING结构域中的突变消除了果糖-1,6-二磷酸酶在体内的多聚泛素化和消除。六种Gid蛋白存在于糖异生细胞中。第七种蛋白Gid4/Vid24在向糖异生细胞中添加葡萄糖后出现，随后被消除。在糖异生细胞中强制异常表达Gid4/Vid24会导致果糖-1,6-二磷酸酶降解。这表明Gid4/Vid24通过Gid复合物启动果糖-1,6-二磷酸酶的多聚泛素化及其随后被蛋白酶体消除。我们还表明，另一种糖异生酶磷酸烯醇式丙酮酸羧激酶也受到Gid复合物依赖性降解的影响。我们的研究揭示了一种新型的泛素连接酶复合物，其由参与碳水化合物代谢的新型亚基组成，并将Gid4/Vid24鉴定为该E3的主要调节因子。
