Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells.
Localization of the Lys, Asp, Glu, Leu tetrapeptide receptor to the Golgi complex and the intermediate compartment in mammalian cells.
复制标题
DOI：
10.1083/jcb.127.6.1557
复制
发表时间：
1994-12
期刊：
影响因子：
--
通讯作者：
Hong W
中科院分区：
文献类型：
--
作者：
Griffiths G;Ericsson M;Krijnse-Locker J;Nilsson T;Goud B;Söling HD;Tang BL;Wong SH;Hong W
羧基末端Lys-Asp-Glu-Leu （KDEL）或一个密切相关的序列，对于管状和II型膜蛋白的内质网定位都很重要。该序列在er后区室中作为检索信号，但检索发生的确切区室仍未确定。利用一种针对哺乳动物KDEL受体羧基末端序列的亲和纯化抗体，我们在不同组织（如小鼠精细胞和大鼠胰腺，以及HeLa、Vero、NRK和小鼠L细胞）的解冻冷冻切片上使用免疫金标记研究了其亚细胞定位。我们发现rab1是中间区室的一个很好的标记，我们使用这个标记，以及感染这种病毒的细胞中的小鼠肝炎病毒（MHV）的出芽谱，来识别这个区室。我们的结果表明，KDEL受体集中在中间室，以及在高尔基堆栈。在粗糙ER中检测到较低但显著的标记。一般来说，只有少量的受体在高尔基体堆栈的反侧被检测到，包括正常细胞和组织的反式高尔基网络（TGN）。然而，一些应激条件，如牛痘病毒或水疱性口炎病毒感染，以及20摄氏度或43摄氏度的处理，导致高尔基体堆栈的分布向反TGN侧显著转移。这种转变可以在稳定表达TGN标记物的HeLa细胞中量化。在所有测试条件下，在TGN远端结构中未检测到显著的标记。在GTP γ S处理过渗透细胞后，在处理后积累的含有β - cop的芽/囊泡中检测到受体，表明这些囊泡可能在隔室之间运输受体。我们认为，在胞吐途径中，含KDEL蛋白的回收发生在多个内质网后室直至TGN，并且在该途径中，不同室中受体的数量根据生理条件而变化。
The carboxyl-terminal Lys-Asp-Glu-Leu (KDEL), or a closely-related sequence, is important for ER localization of both lumenal as well as type II membrane proteins. This sequence functions as a retrieval signal at post-ER compartment(s), but the exact compartment(s) where the retrieval occurs remains unresolved. With an affinity-purified antibody against the carboxyl-terminal sequence of the mammalian KDEL receptor, we have investigated its subcellular localization using immunogold labeling on thawed cryosections of different tissues, such as mouse spermatids and rat pancreas, as well as HeLa, Vero, NRK, and mouse L cells. We show that rab1 is an excellent marker of the intermediate compartment, and we use this marker, as well as budding profiles of the mouse hepatitis virus (MHV) in cells infected with this virus, to identify this compartment. Our results demonstrate that the KDEL receptor is concentrated in the intermediate compartment, as well as in the Golgi stack. Lower but significant labeling was detected in the rough ER. In general, only small amounts of the receptor were detected on the trans side of the Golgi stack, including the trans- Golgi network (TGN) of normal cells and tissues. However, some stress conditions, such as infection with vaccinia virus or vesicular stomatitis virus, as well as 20 degrees C or 43 degrees C treatment, resulted in a significant shift of the distribution towards the trans- TGN side of the Golgi stack. This shift could be quantified in HeLa cells stably expressing a TGN marker. No significant labeling was detected in structures distal to the TGN under all conditions tested. After GTP gamma S treatment of permeabilized cells, the receptor was detected in the beta-COP-containing buds/vesicles that accumulate after this treatment, suggesting that these vesicles may transport the receptor between compartments. We propose that retrieval of KDEL- containing proteins occurs at multiple post-ER compartments up to the TGN along the exocytotic pathway, and that within this pathway, the amounts of the receptor in different compartments varies according to physiological conditions.