Maegawa Saki, Ito Koreaki, Akiyama Yoshinori
Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan.
Biochemistry. 2005 Oct 18;44(41):13543-52. doi: 10.1021/bi051363k.
We characterized Escherichia coli GlpG as a membrane-embedded protease and a possible player in the regulated intramembrane proteolysis in this organism. From the sequence features, it belongs to the widely conserved rhomboid family of membrane proteases. We verified the expected topology of GlpG, and it traverses the membrane six times. A model protein having an N-terminal and periplasmically localized beta-lactamase (Bla) domain, a LacY-derived transmembrane region, and a cytosolic maltose binding protein (MBP) mature domain was found to be GlpG-dependently cleaved in vivo. This proteolytic reaction was reproduced in vitro using purified GlpG and purified model substrate protein, and the cleavage was shown to occur between Ser and Asp in a region of high local hydrophilicity, which might be located in a juxtamembrane rather than an intramembrane position. The conserved Ser and His residues of GlpG were essential for the proteolytic activities. Our results using several variant forms of the model protein suggest that GlpG recognizes features of the transmembrane regions of substrates. These results point to a detailed molecular mechanism and cellular analysis of this interesting class of membrane-embedded proteases.
我们将大肠杆菌GlpG鉴定为一种膜嵌入蛋白酶，它可能参与了该生物体的调节性膜内蛋白水解过程。从序列特征来看，它属于广泛保守的类菱形膜蛋白酶家族。我们验证了GlpG预期的拓扑结构，它跨膜六次。发现一种具有N端和周质定位的β-内酰胺酶（Bla）结构域、LacY衍生的跨膜区域以及胞质麦芽糖结合蛋白（MBP）成熟结构域的模型蛋白在体内可被GlpG依赖性切割。使用纯化的GlpG和纯化的模型底物蛋白在体外重现了这种蛋白水解反应，并且显示切割发生在高局部亲水性区域的丝氨酸和天冬氨酸之间，该区域可能位于近膜而非膜内位置。GlpG保守的丝氨酸和组氨酸残基对蛋白水解活性至关重要。我们使用该模型蛋白几种变体形式的结果表明，GlpG识别底物跨膜区域的特征。这些结果指向了这类有趣的膜嵌入蛋白酶的详细分子机制和细胞分析。
