Glycosylation's active site—finally
http://www.100md.com
《细胞学杂志》
Beginning on page 715, Nilsson et al. drive home the lesson that persistence pays. Using established techniques on a system that has frustrated similar efforts for years, the authors identified the active site of the mammalian oligosaccharyltransferase (OST) complex. In the endoplasmic reticulum, OST carries out N-linked glycosylation, one of the most common and least understood eukaryotic protein modifications. Previous efforts to identify the active site of OST using pepftide substrates, or even to determine which of the proteins in the OST complex contains the active site, have produced conflicting results.
The authors designed nascent polypeptide chains with cryptic glycosylation sites incorporating photoreactive probes. As the cryptic site translocates through the endoplasmic reticulum membrane, it can be cross-linked first to components of the translocon pore, and then to the OST. Strikingly, only one OST protein, STT3, is cross-linked in this way, providing strong evidence that the nascent chain portion of the OST active site lies largely or entirely within STT3. Probes placed immediately adjacent to the cryptic glycosylation sequence did not cross-link any OST components. Thus, the high specificity of OST for the glycosylation sequence, and the short residence time of incorrect and glycosylated sequences in the active site, probably doomed earlier efforts to cross-link nascent chains to OST.(STT3 (asterisk) is cross-linked to crypt)
The authors designed nascent polypeptide chains with cryptic glycosylation sites incorporating photoreactive probes. As the cryptic site translocates through the endoplasmic reticulum membrane, it can be cross-linked first to components of the translocon pore, and then to the OST. Strikingly, only one OST protein, STT3, is cross-linked in this way, providing strong evidence that the nascent chain portion of the OST active site lies largely or entirely within STT3. Probes placed immediately adjacent to the cryptic glycosylation sequence did not cross-link any OST components. Thus, the high specificity of OST for the glycosylation sequence, and the short residence time of incorrect and glycosylated sequences in the active site, probably doomed earlier efforts to cross-link nascent chains to OST.(STT3 (asterisk) is cross-linked to crypt)