Ivessa N E, De Lemos-Chiarandini C, Gravotta D, Sabatini D D, Kreibich G
New York University School of Medicine, New York, USA.
J Biol Chem. 1995 Oct 27;270(43):25960-7. doi: 10.1074/jbc.270.43.25960.
Ribophorin I is a type I transmembrane glycoprotein specific to the rough endoplasmic reticulum. We have previously shown that, when expressed in transfected HeLa cells, a carboxyl-terminally truncated form of ribophorin I that contains most of the luminal domain (RI332) is, like the native protein, retained in the endoplasmic reticulum (ER). Brefeldin A (BFA) treatment of these HeLa cells leads to O-glycosylation of RI332 by glycosyltransferases that are redistributed from the Golgi apparatus to the ER (Ivessa, N. E., De Lemos-Chiarandini, C., Tsao, Y.-S., Takatsuki, A., Adesnik, M., Sabatini, D. D., and Kreibich, G. (1992) J. Cell Biol. 117, 949-958). Using the state of glycosylation of RI332 as a measure for the BFA-induced backflow of enzymes of the Golgi apparatus to the ER, we now demonstrate that the retrograde transport is inhibited when cells are treated with various agents that affect intracellular Ca2+ concentrations, such as the dipeptide benzyloxycarbonyl (Cbz)-Gly-Phe-amide, the Ca2+ ionophore A23187, and thapsigargin, an inhibitor of the Ca(2+)-transporting ATPase of the ER. These treatments prevent the BFA-induced O-glycosylation of RI332. Immunofluorescence localization of the Golgi markers, MG-160 and galactosyltransferase, shows that when BFA is applied in the presence of Ca2+ modulating agents, the markers remain confined to the Golgi apparatus and are not redistributed to the ER, as is the case when BFA alone is used. Cbz-Gly-Phe-amide does not, however, interfere with the BFA-induced release of beta-COP from the Golgi apparatus. We conclude that the maintenance of a Ca2+ gradient between the cytoplasm and the lumen of the ER and the Golgi apparatus is required for the BFA-induced retrograde transport from the Golgi apparatus to the ER to occur.
核糖体结合蛋白I是一种糙面内质网特有的I型跨膜糖蛋白。我们之前已经表明，当在转染的HeLa细胞中表达时，一种羧基末端截短但包含大部分腔结构域的核糖体结合蛋白I（RI332），与天然蛋白一样，保留在内质网（ER）中。用布雷菲德菌素A（BFA）处理这些HeLa细胞会导致RI332被从高尔基体重新分布到内质网的糖基转移酶进行O-糖基化（伊韦萨，N.E.，德莱莫斯-基亚兰迪尼，C.，曹，Y.-S.，高槻，A.，阿德尼克，M.，萨巴蒂尼，D.D.，和克雷比希，G.（1992年）《细胞生物学杂志》117卷，949 - 958页）。利用RI332的糖基化状态作为衡量BFA诱导的高尔基体酶回流到内质网的指标，我们现在证明，当用各种影响细胞内Ca2+浓度的试剂处理细胞时，逆行转运受到抑制，这些试剂如二肽苄氧羰基（Cbz）-甘氨酰-苯丙氨酸酰胺、Ca2+离子载体A-二甲基亚砜、A23187和毒胡萝卜素，后者是内质网Ca(2+)-转运ATP酶的抑制剂。这些处理可防止BFA诱导的RI332的O-糖基化。高尔基体标志物MG-160和半乳糖基转移酶的免疫荧光定位表明，当在Ca2+调节剂存在的情况下应用BFA时，标志物仍局限于高尔基体，不会像单独使用BFA时那样重新分布到内质网。然而，Cbz-甘氨酰-苯丙氨酸酰胺并不干扰BFA诱导的β-COP从高尔基体释放。我们得出结论，内质网和高尔基体腔与细胞质之间Ca2+梯度的维持是BFA诱导的从高尔基体到内质网逆行转运发生所必需的。
