FIGURE 5.

MPEG-5 reactivity with cysteine mutants. A, a schematic of trypsin fragment V with the cytosolic-facing 13 endogenous cysteines potentially accessible to MPEG-5 (orange) and seven inaccessible endogenous cysteines (purple). The central panel of blots (B) shows the effect of deleting segments of the C-terminal tail corresponding to the removal of 24 (TL-1), 60 (TL-2), and 141 (TL-5) amino acids. The latter construct removes the three endogenous cysteines in the C-tail. Each of the tail-less constructs contained a C-terminal HA-tag for detection. Microsomal membranes were prepared and incubated in the presence or absence of 2.2 μm free [Ca²⁺] for 5 min followed by a 5-min incubation with 0.5 mm MPEG-5 (lanes 3 and 4). All samples were processed on 7% SDS-PAGE. Unshifted and shifted bands (open and closed triangles, respectively) were detected by immunoblotting with IL-3 or HA antibodies. The sequences in C are derived from the C-terminal region of the receptor and indicate potential trypsin cleavage sites (underlined italic) preceding the CT-1 Ab epitope (pink line). The blots in D show the effect of mutating each of the 10 endogenous cysteines in the segment prior to the transmembrane domains. The highlighted panel shows the elimination of the MPEG shift upon mutating a cluster of six closely spaced cysteines. The experimental conditions are as described for B except IL-3 Ab was used for detection of all blots. ER, endoplasmic reticulum.