Pirjo M. Apaja, Haijin Xu, Gergely L. Lukacs (2011) CIL:13685, 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 non-permeabilized COS7 cell and the apparent plasma membrane targeting shows the value of this model plasma membrane protein. The ER marker, calreticulin antibody (red), is not apparent here because lack of detergent-permeabilization limited access of the antibody to the ER.

为探究质膜蛋白错折叠的细胞学后果，开发了一种组成型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端截短的CD4（CD4tl）构成，并融合到野生型（wt）噬菌体λ抑制剂的N端DNA结合域（CD4tl-lambda）或L57C突变抑制剂的N端DNA结合域（CD4tl-lambdaC）。在26°C时，CD4tl-lambdaC的细胞质域主要处于天然状态，而在37°C时则主要处于非天然状态，从而允许利用热变性追踪未折叠蛋白的命运。在本图像中，展示了Pirjo等人（JCB 2010年第1图的六组之一）中控制报告分子CD4tl-lambda在37°C的表达情况，使用CD4抗体（绿色）在未渗透化的COS7细胞中观察到，其明显的质膜靶向展示了该模型质膜蛋白的价值。由于缺乏去污剂渗透，ER标记物钙网蛋白抗体（红色）在此不显著。