Grab D J, Webster P, Verjee Y
Proc Natl Acad Sci U S A. 1984 Dec;81(24):7703-7. doi: 10.1073/pnas.81.24.7703.
Pulse-chase experiments using L-[35S]methionine suggest that Trypanosoma brucei MITat 1.2 variable surface glycoprotein (VSG) synthesized in the rough endoplasmic reticulum, a process that takes 6-8 min, is shuttled to the Golgi complex 8 min later. Labeling of ultrathin frozen sections with affinity-purified anti-cross-reacting determinant (CRD) IgG followed by protein A-colloidal gold shows that the CRD is localized in the trans-Golgi region. cis-Golgi is not labeled. VSG, when solubilized by treatment with the detergent Nonidet P-40, behaves on sucrose density gradients as a non-membrane protein with a sedimentation value of 5 S. In contrast, VSG solubilized in the presence of Zwittergent TM 3-14 yielded several VSG-containing fractions greater than 5 S, and only the 5S fraction contained the CRD. Lack of the CRD in VSG complexes with sedimentation values greater than 5 S suggests that this determinant is either masked from antibody, perhaps by involvement in polymer formation, or represents the membrane form of VSG recently described by Cardoso de Almeida and Turner [Cardoso de Almeida, M. L. & Turner, M. J. (1983) Nature (London) 302, 349-352].
使用L-[35S]甲硫氨酸进行的脉冲追踪实验表明，布氏锥虫MITat 1.2可变表面糖蛋白（VSG）在糙面内质网中合成，这一过程需要6 - 8分钟，8分钟后被转运至高尔基体复合体。用亲和纯化的抗交叉反应决定簇（CRD）IgG标记超薄冷冻切片，随后用蛋白A - 胶体金标记，结果显示CRD定位于反式高尔基体区域。顺式高尔基体未被标记。当用去污剂Nonidet P - 40处理使VSG溶解时，它在蔗糖密度梯度上表现为沉降值为5 S的非膜蛋白。相比之下，在两性离子去污剂TM 3 - 14存在下溶解的VSG产生了几个沉降值大于5 S的含VSG的组分，并且只有5 S组分含有CRD。沉降值大于5 S的VSG复合物中缺乏CRD，这表明该决定簇要么被抗体掩盖，可能是由于参与了聚合物形成，要么代表了最近由卡多索·德阿尔梅达和特纳描述的VSG的膜形式[卡多索·德阿尔梅达，M. L. & 特纳，M. J.（1983年）《自然》（伦敦）302，349 - 352]。
