Audigier Y, Nigam S K, Blobel G
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.
J Biol Chem. 1988 Nov 5;263(31):16352-7.
Employing [32P]ADP-ribosylation by pertussis toxin we have identified a G protein that is located in the rough endoplasmic reticulum of canine pancreas and therefore termed it GRER. Identification of GRER is based on the following data. A 41-kDa polypeptide was the only polypeptide that was [32P]ADP-ribosylated by pertussis toxin in pancreas rough microsomes. Guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) and 1 mM ATP, 6 mM MgCl2, 10 mM NaF (AMF) inhibited ADP-ribosylation of this polypeptide. The [32P]ADP-ribosylated 41-kDa polypeptide was immunoprecipitated by antisera which specifically recognized the C-terminal residues of the alpha subunits of Gi and transducin, indicating that the 41-kDa polypeptide is immunologically related to the alpha subunits of heterotrimeric G proteins. Treatment with GTP gamma S resulted in a reduction in the sedimentation rate of the [32P]ADP-ribosylated, detergent-solubilized GRER. It also induced the release of the [32P]ADP-ribosylated 41-kDa polypeptide from rough microsomes in the absence of detergent, unlike ADP-ribosylated alpha subunits of plasma membrane-associated G proteins. These data are consistent with an oligomeric nature of GRER. The codistribution of GRER with an endoplasmic reticulum marker protein during subcellular fractionation and the lack of plasma membrane contamination of the rough microsomal fraction, combined with the isodensity of GRER with rough microsomes as well as the isodensity of GRER with "stripped" microsomes after extraction of rough microsomes with EDTA and 0.5 M KCl, localized GRER to the rough endoplasmic reticulum. Preliminary experiments suggest that GRER appears not to be involved in translocation of proteins across the rough endoplasmic reticulum membrane.
利用百日咳毒素进行的[32P]ADP-核糖基化反应，我们鉴定出一种位于犬胰腺粗面内质网中的G蛋白，因此将其命名为GRER。GRER的鉴定基于以下数据。一种41 kDa的多肽是胰腺粗面微粒体中唯一被百日咳毒素进行[32P]ADP-核糖基化的多肽。鸟苷5'-(γ-硫代)三磷酸(GTPγS)以及1 mM ATP、6 mM MgCl2、10 mM NaF(AMF)可抑制该多肽的ADP-核糖基化。[32P]ADP-核糖基化的41 kDa多肽被能特异性识别Gi和转导素α亚基C末端残基的抗血清免疫沉淀，这表明该41 kDa多肽在免疫上与异源三聚体G蛋白的α亚基相关。用GTPγS处理导致[32P]ADP-核糖基化的、经去污剂溶解的GRER沉降速率降低。与质膜相关G蛋白的ADP-核糖基化α亚基不同，它还能在无去污剂的情况下诱导[32P]ADP-核糖基化的41 kDa多肽从粗面微粒体中释放。这些数据与GRER的寡聚性质一致。在亚细胞分级分离过程中，GRER与内质网标记蛋白的共分布以及粗面微粒体部分不存在质膜污染，再加上GRER与粗面微粒体的等密度以及用EDTA和0.5 M KCl提取粗面微粒体后GRER与“脱蛋白”微粒体的等密度，将GRER定位到粗面内质网。初步实验表明，GRER似乎不参与蛋白质跨粗面内质网膜的转运。
