Tanaka Satoshi, Kinoshita Jun-ya, Kuroda Rieko, Ito Akio
Department of Chemistry, Faculty of Science, Kyushu University, Fukuoka 812-8581, Japan.
J Biochem. 2003 Feb;133(2):247-51. doi: 10.1093/jb/mvg034.
Integration of cytochrome b(5) (b5), a tail-anchored protein located in the endoplasmic reticulum (ER) membrane, into the membrane was studied. Mutation of three amino acids, -Leu-Met-Tyr, at the carboxy-terminal end of the transmembrane segment of b5 to alanines resulted in localization of the mutated protein, b5LMY/AAA, in the cytosol as well as in the ER membrane. When an N-glycosylation site was introduced at the carboxy-terminal end of b5LMY/AAA, a substantial amount of the glycosylated form of the mutant protein was recovered in the cytosol fraction. A portion of the mutant protein recovered in the ER was released from the membrane by incubation with the cytosol fraction, but no further release was observed in the second incubation, suggesting that b5 is present in two different states, loosely-bound and firmly-integrated forms, in the ER membrane. These results suggest that b5 is integrated into the ER membrane via the loosely bound state, in which the carboxy-terminal end of the molecule is inserted into the luminal side of the vesicle but is easily translocated back to the cytosol, and that the three amino acids are important for conversion of the loosely-bound state to the firmly-integrated state.
研究了细胞色素b(5)（b5），一种位于内质网（ER）膜上的尾锚定蛋白，整合到膜中的过程。b5跨膜区段羧基末端的三个氨基酸-Leu-Met-Tyr突变为丙氨酸，导致突变蛋白b5LMY/AAA定位于细胞质以及ER膜中。当在b5LMY/AAA的羧基末端引入一个N-糖基化位点时，在细胞质组分中回收了大量糖基化形式的突变蛋白。在ER中回收的一部分突变蛋白通过与细胞质组分一起孵育而从膜上释放，但在第二次孵育中未观察到进一步释放，这表明b5在ER膜中以两种不同状态存在，即松散结合和牢固整合形式。这些结果表明，b5通过松散结合状态整合到ER膜中，在这种状态下，分子的羧基末端插入囊泡的腔侧，但很容易转运回细胞质，并且这三个氨基酸对于从松散结合状态转变为牢固整合状态很重要。
