Rodriguez-Cousiño N, Nargang F E, Baardman R, Neupert W, Lill R, Court D A
Adolf Butenandt Institut für Physiologische Chemie, Ludwig-Maximilians-Universität München, Goethestrasse 33, 80336 München, Germany.
J Biol Chem. 1998 May 8;273(19):11527-32. doi: 10.1074/jbc.273.19.11527.
TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The protein is anchored to the lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The orientation of the protein is not influenced by the charges flanking its trans-membrane segment, in contrast to observations regarding proteins of other membranes. In vitro import studies utilizing TOM22 preproteins harboring deletions or mutations in the cytosolic domain revealed that the combination of the trans-membrane segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment of the cytosolic domain was found to be essential for the import and assembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in which the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 contains physically separate import and membrane anchor sequences.
TOM22是线粒体外膜前体蛋白转位酶（TOM复合体）的一个组成部分。该蛋白通过一个中央跨膜片段锚定在脂质双层上，从而使氨基末端结构域暴露于细胞质溶胶中，羧基末端部分暴露于膜间隙中。在此，我们描述了将粗糙脉孢菌TOM22靶向并正确插入外膜的序列要求。与其他膜蛋白的观察结果相反，该蛋白的方向不受其跨膜片段两侧电荷的影响。利用在细胞质结构域中含有缺失或突变的TOM22前体蛋白进行的体外导入研究表明，TOM22的跨膜片段和膜间隙结构域的组合不足以指导其导入外膜。相反，发现细胞质结构域的一小段对于TOM22的导入和组装至关重要。这个序列是外膜的一种新型内部导入信号，带有净正电荷。导入信号电荷改变为-1的突变型TOM22的导入效率低于野生型蛋白。我们的数据表明，TOM22包含物理上分开的导入序列和膜锚定序列。
