GOLGI RETENTION OF A TRANS-GOLGI MEMBRANE-PROTEIN, GALACTOSYL-TRANSFERASE, REQUIRES CYSTEINE AND HISTIDINE-RESIDUES WITHIN THE MEMBRANE-ANCHORING DOMAIN
GOLGI RETENTION OF A TRANS-GOLGI MEMBRANE-PROTEIN, GALACTOSYL-TRANSFERASE, REQUIRES CYSTEINE AND HISTIDINE-RESIDUES WITHIN THE MEMBRANE-ANCHORING DOMAIN
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DOI：
10.1073/pnas.89.10.4319
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发表时间：
1992-05-15
影响因子：
11.1
通讯作者：
FUKUDA, MN
中科院分区：
文献类型：
--
作者：
AOKI, D;LEE, N;FUKUDA, MN
半乳糖基转移酶（GT; UDP半乳糖：β-D-N-乙酰氨基葡糖苷β-1，4-半乳糖基转移酶，EC 2.4.1.22）是一种II型膜锚定蛋白，由短的N-末端胞质尾、信号/膜锚定结构域和茎区以及随后的包括C末端的大催化结构域组成。为了鉴定GT高尔基体定位的关键肽段和关键氨基酸残基，设计表达载体pGT-hCG以编码与人绒毛膜促性腺激素α亚基（hCG-α）的C-末端区域融合的整个GT分子作为报告基因。转染pGT.hCG的COS-1细胞在高尔基体区域表达嵌合体，用抗hCG抗体通过免疫荧光显微镜检测。两个缺失突变体，DELTA尾和DELTA茎，这是缺乏大部分的N-末端胞质尾或10个氨基酸后立即膜锚定结构域，定位在高尔基体。GT的膜锚定结构域的置换突变表明，跨膜结构域的第二个四分之一或Cys 29-Ala 30-Leu 31-His 32-Leu 33是GT保留在高尔基体所必需的。此外，点突变体Cys 29-> Ser 29和His 32-> Leu 32部分转运到质膜，而Ala 30-Leu 31-> Phe 30-Gly 31突变体定位于高尔基体。最后，发现一个双突变体，Cys 29/His 32--> Ser 29/Leu 32，被有效地运输到质膜。转铁蛋白受体的信号锚定结构域，II型质膜蛋白，然后被GT跨膜结构域的部分取代。虽然Cys-Xaa-Xaa-His序列本身不能保留高尔基体中的转铁蛋白受体，但GT的跨膜结构域的胞质半部分能够保留高尔基体中的转铁蛋白受体。这些结果表明，GT的跨膜结构域的胞质（或N-末端）的一半有助于高尔基体保留信号，特别是该区域中的Cys 29和His 32是GT保留在高尔基体中的关键。
Galactosyltransferase (GT; UDPgalactose: beta-D-N-acetylglucosaminide beta-1,4-galactosyltransferase, EC 2.4.1.22) is a type II membrane-anchored protein composed of a short N-terminal cytoplasmic tail, a signal/membrane-anchoring domain, and a stem region followed by a large catalytic domain including the C terminus. To identify the peptide segment and key amino acid residues that are critical for Golgi localization of GT, the expression vector pGT-hCG was designed to encode the entire GT molecule fused to the C-terminal region of human chorionic gonadotropin alpha-subunit (hCG-alpha) as a reporter. COS-1 cells transfected with pGT.hCG expressed the chimera in the Golgi region, as detected by immunofluorescence microscopy using anti-hCG antibodies. Two deletion mutants, DELTA-tail and DELTA-stem, which are lacking most of the N-terminal cytoplasmic tail or 10 amino acids immediately after the membrane-anchoring domain, were localized in the Golgi. Replacement mutations of the membrane-anchoring domain of GT showed that the second quarter of the transmembrane domain or Cys29-Ala30-Leu31-His32-Leu33 is necessary for GT to be retained in the Golgi. Furthermore, the point mutants Cys29 --> Ser29 and His32 --> Leu32 were partially transported to the plasma membrane, whereas an Ala30-Leu31 --> Phe30-Gly31 mutant was localized in the Golgi. Finally, a double mutant, Cys29/His32 --> Ser29/Leu32, was found to be transported efficiently to the plasma membrane. The signal-anchoring domain of the transferrin receptor, a type II plasma membrane protein, was then replaced by portions of the GT transmembrane domain. Although the Cys-Xaa-Xaa-His sequence by itself cannot retain the transferrin receptor in the Golgi, the cytoplasmic half of the transmembrane domain of GT was partially capable of retaining the transferrin receptor in the Golgi. These results suggest that the cytoplasmic (or N-terminal) half of the transmembrane domain of GT contributes to the Golgi retention signal and that particularly Cys29 and His32 in this region are critical for GT to be retained in the Golgi.