Nilsson T, Lucocq J M, Mackay D, Warren G
Cell Biology Laboratory, Imperial Cancer Research Fund, London, UK.
EMBO J. 1991 Dec;10(12):3567-75. doi: 10.1002/j.1460-2075.1991.tb04923.x.
Chimeric cDNAs were constructed so as to generate hybrid proteins in which different parts of the N-terminal domain of the human invariant chain were replaced by equivalent sequences from the trans Golgi resident enzyme, beta-1,4-galactosyltransferase. The cytoplasmic and membrane spanning domains of galactosyltransferase were found to be sufficient to retain all of the hybrid invariant chain in trans Golgi cisternae as judged by indirect immunofluorescence, treatment with brefeldin A and immuno-electron microscopy. As few as ten amino acids corresponding to the lumenal half of the membrane spanning domain of the Golgi enzyme sufficed to localize most of the hybrid invariant chain to the trans cisternae. A cytoplasmic domain was necessary for complete retention as assessed by flow cytofluorometry but could be provided either by galactosyltransferase or by invariant chain. This suggests that the cytoplasmic domain plays a role accessory to the membrane spanning domain, the latter mediating compartmental specificity.
构建嵌合cDNA以产生杂合蛋白，其中人恒定链N端结构域的不同部分被反式高尔基体驻留酶β-1,4-半乳糖基转移酶的等效序列取代。通过间接免疫荧光、布雷菲德菌素A处理和免疫电子显微镜判断，发现半乳糖基转移酶的细胞质和跨膜结构域足以将所有杂合恒定链保留在反式高尔基体潴泡中。与高尔基体酶跨膜结构域腔侧一半相对应的仅十个氨基酸就足以将大部分杂合恒定链定位到反式潴泡中。通过流式细胞荧光术评估，细胞质结构域对于完全保留是必需的，但可以由半乳糖基转移酶或恒定链提供。这表明细胞质结构域发挥的作用是辅助跨膜结构域的，后者介导区室特异性。
