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 26 degrees is seen with CD4 antibody (green) in a non-permeabilized COS7 cell. At this temperature, the folding and processing of this mutant membrane reporter allows 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.
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