Pirjo M. Apaja, Haijin Xu, Gergely L. Lukacs (2011) CIL:13681, 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 mutant plasma membrane reporter, CD4tl-lambdaC, at 37 degrees is seen with CD4 antibody (green) in a detergent-permeabilized COS7 cell. The intracellular processing of this nonnative, thermally unfolded protein nearly completely limits its targeting to the plasma membrane. This cell is co-labeled with an ER marker, calreticulin antibody (red).

为探究质膜蛋白错折叠的细胞学后果，开发了一种常驻靶向质膜的跨膜模型蛋白，并用作报告分子。该报告分子由C端截断的CD4（CD4tl）构成，其中融入了灵活的细胞质连接器，并与野生型（wt）噬菌体λ抑制剂的N端DNA结合域融合，形成CD4tl-lambda；或与L57C突变抑制剂的N端DNA结合域融合，形成CD4tl-lambdaC。在26°C下，CD4tl-lambdaC的细胞质域主要处于天然状态，而在37°C下则主要处于非天然状态，从而允许利用热变位追踪非折叠蛋白的命运。在本图像中，可见于Pirjo等人在2010年JCB杂志第1图的六组之一，展示了一种突变质膜报告分子CD4tl-lambdaC在37°C下的表达，使用CD4抗体（绿色）在去污剂透化后的COS7细胞中进行标记。这种非天然、热变性的蛋白在细胞内处理过程中几乎完全限制了其靶向质膜的能力。该细胞同时标记了内质网标记物，即钙网蛋白抗体（红色）。