Teasdale R D, D'Agostaro G, Gleeson P A
Department of Pathology and Immunology, Monash University Medical School, Melbourne, Victoria, Australia.
J Biol Chem. 1992 Feb 25;267(6):4084-96.
The expression and localization of bovine beta 1,4-galactosyltransferase (Gal T) has been studied in mammalian cells transfected with Gal T cDNA constructs, and the role of the amino-terminal domains of Gal T in Golgi localization examined. Here we demonstrate that the transmembrane (signal/anchor) domain of bovine Gal T contains a positive Golgi retention signal. Bovine Gal T was characterized in transfected cells with anti-bovine Gal T antibodies, affinity-purified from a rabbit antiserum using a bacterial recombinant fusion protein. These affinity-purified antibodies recognized native bovine Gal T and showed minimum cross-reactivity with Gal T from non-bovine sources. Bovine Gal T cDNA was expressed, as active enzyme, transiently in COS-1 cells and stably in murine L cells, and the product was shown to be localized to the Golgi complex by immunofluorescence using the polypeptide-specific antibodies. A low level of surface bovine Gal T was also detected in the transfected L cells by flow cytometry. The removal of 18 of the 24 amino acids from the cytoplasmic domain of bovine Gal T did not alter the Golgi localization of the product transiently expressed in COS-1 cells or stably expressed in L cells. Both the full-length bovine Gal T and the cytoplasmic domain deletion mutant were N-glycosylated in the transfected L cells, indicating both proteins have the correct N(in)/C(out) membrane orientation. Deletion of both the cytoplasmic and signal/anchor domains of bovine Gal T and incorporation of a cleavable signal sequence resulted in a truncated soluble bovine Gal T that was rapidly secreted (within 1 h) from transfected COS-1 cells. Replacement of the signal/anchor domain of bovine Gal T with the signal/anchor domain of the human transferrin receptor resulted in the transport of the hybrid molecule to the cell surface of transfected COS-1 cells. Furthermore, a hybrid construct containing the signal/anchor domain of Gal T with ovalbumin was efficiently retained in the Golgi complex, whereas ovalbumin anchored to the membrane by the transferrin receptor signal/anchor was expressed at the cell surface of transfected COS-1 cells. Overall, these studies show that the hydrophobic, signal/anchor domain of Gal T is both necessary and sufficient for Golgi localization.
在转染了半乳糖基转移酶（Gal T）cDNA构建体的哺乳动物细胞中，研究了牛β1,4 - 半乳糖基转移酶（Gal T）的表达和定位，并检测了Gal T氨基末端结构域在高尔基体定位中的作用。在此，我们证明牛Gal T的跨膜（信号/锚定）结构域包含一个正向高尔基体保留信号。使用抗牛Gal T抗体对转染细胞中的牛Gal T进行了表征，该抗体是从兔抗血清中通过细菌重组融合蛋白亲和纯化得到的。这些亲和纯化的抗体识别天然牛Gal T，并且与非牛来源的Gal T交叉反应性最小。牛Gal T cDNA在COS - 1细胞中瞬时表达为活性酶，在小鼠L细胞中稳定表达，使用多肽特异性抗体通过免疫荧光显示产物定位于高尔基体复合体。通过流式细胞术在转染的L细胞中也检测到了低水平的表面牛Gal T。从牛Gal T的胞质结构域去除24个氨基酸中的18个，并未改变在COS - 1细胞中瞬时表达或在L细胞中稳定表达的产物的高尔基体定位。全长牛Gal T和胞质结构域缺失突变体在转染的L细胞中均进行了N - 糖基化，表明这两种蛋白都具有正确的N（内）/C（外）膜取向。删除牛Gal T的胞质和信号/锚定结构域并掺入可裂解信号序列，导致截短的可溶性牛Gal T从转染的COS - 1细胞中迅速分泌（在1小时内）。用人转铁蛋白受体的信号/锚定结构域替换牛Gal T的信号/锚定结构域，导致杂合分子转运到转染的COS - 1细胞的细胞表面。此外，包含Gal T信号/锚定结构域与卵清蛋白的杂合构建体有效地保留在高尔基体复合体中，而通过转铁蛋白受体信号/锚定锚定在膜上的卵清蛋白在转染的COS - 1细胞的细胞表面表达。总体而言，这些研究表明Gal T的疏水信号/锚定结构域对于高尔基体定位既必要又充分。
