Pirjo M. Apaja, Haijin Xu, Gergely L. Lukacs (2011) CIL:13684, Chlorocebus aethiops. CIL. Dataset

To study the cellular consequences of misfolding of plasma membrane proteins, a transmembrane model protein that was constitutively targeted to the plasma membrane was developed for use as a reporter molecule. The reporter consisted of a C-terminally truncated CD4, incorporating a flexible cytoplasmic linker (CD4tl), fused to the N-terminal DNA-binding domain of the wild type (wt) bacteriophage lambda repressor (CD4tl-lambda) or L57C mutant repressor (CD4tl-lambdaC). The CD4tl-lambdaC cytosolic domain was largely in native state at 26°C but predominantly nonnative state at 37°C thus allowing the use of thermal shifts to follow the fate of unfolded proteins. In this image, one of six of a group from Figure 1 of Pirjo et al., JCB 2010, the expression of the control reporter, CD4tl-lambda, at 37 degrees is seen with CD4 antibody (green) in a detergent-permeabilized COS7 cell and the apparent plasma membrane targeting shows the value of this model plasma membrane protein. The cell is co-labeled for an ER marker, calreticulin antibody (red)..

为探究质膜蛋白错误折叠的细胞学后果，开发了一种常驻靶向质膜的跨膜模型蛋白，并将其用作报告分子。该报告分子由C端截断的CD4（CD4tl）构成，其中包含一个灵活的细胞质连接区（CD4tl），与野生型（wt）噬菌体λ阻遏蛋白的N端DNA结合域融合（CD4tl-lambda）或L57C突变阻遏蛋白（CD4tl-lambdaC）。在26°C下，CD4tl-lambdaC的细胞质结构域主要处于天然状态，而在37°C下则主要处于非天然状态，从而允许利用热变性技术追踪非折叠蛋白的命运。在本图像中，可见自Pirjo等人2010年JCB杂志图1中的六个分组之一，在37°C下控制报告分子CD4tl-lambda的表达，使用CD4抗体（绿色）在去污剂渗透性COS7细胞中，其明显的质膜靶向展示了该模型质膜蛋白的价值。细胞同时标记了内质网标记物，即钙网蛋白抗体（红色）。