FIGURE 1:

In vitro synthesis and degradation of CFTR. (A) Schematic representation of CFTR showing membrane topology and approximate location of endogenous methionine residues. (B) Phosphorimage of CFTR translated in RRL in the presence of canine pancreas rough microsomes and [³⁵S]Met. Aliquots were taken at indicated times and analyzed by 7% SDS–PAGE and phosphorimaging. Migration of full-length CFTR protein is indicated. (C) Microsomes containing [³⁵S]-labeled CFTR were collected and incubated at 37°C in RRL (minus hemin) in the presence of 100 μM MG132 (+MG132) or after ATP depletion by hexokinase and 2-deoxyglucose (–ATP). Samples were analyzed by SDS–PAGE and phosphorimaging at times indicated. (D) Aliquots of degradation reaction were precipitated by TCA and analyzed by scintillation counting. The percentage of CFTR degraded into TCA-soluble fragments at each point is shown as mean ± SEM (n = 3).