Pirjo M. Apaja, Haijin Xu, Gergely L. Lukacs (2011) CIL:13683, 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 non-permeabilized COS7 cells. The intracellular processing of this nonnative, thermally unfolded protein nearly completely limits its targeting to the plasma membrane. The ER marker, calreticulin antibody (red), is not apparent here because lack of detergent-permeabilization limited access of the antibody to the ER.

为研究质膜蛋白质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，内嵌一个灵活的细胞质连接域，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，因此允许利用热变位法追踪未折叠蛋白质的命运。在本图中，展示了Pirjo等人在JCB 2010年发表的图1中六个分组之一，在37°C下，使用CD4抗体（绿色）在未经渗透化的COS7细胞中观察到了突变质膜报告分子CD4tl-lambdaC的表达。这种非天然、热变性的蛋白质在细胞内加工过程中几乎完全限制了其靶向质膜的能力。由于缺乏洗涤剂渗透，内质网标记物——钙网蛋白抗体（红色）在此处并不明显。