FIG. 2.

Analysis of Gag-Gag interactions by FRET fluorometry. Membrane and cytosolic fractions were collected for each sample as described in Materials and Methods. Both fractions were analyzed in a scanning cuvette fluorometer. (A) Gag-CFP-Gag-YFP FRET curve for the membrane fraction (closed diamonds) and the cytosolic fraction (open diamonds). The YFP emission peak at 527 nm is indicated by the arrow. Squares, Gag-YFP alone; triangles, Gag-CFP alone. Gag-YFP coexpressed with pECFP-f is also shown for membrane (×) and cytosolic (+) fractions. (B) Relative levels of cellular YFP expression are shown for the experiment depicted in panel A, as determined by peak YFP output following excitation of cell lysates at 514 nm. (C) Wild-type Gag is able to efficiently interact with Myr⁻ Gag in the membrane fraction (closed diamonds and triangles) but not in the cytosolic fraction (open diamonds and triangles). The rest of the symbols are as for panel A. (D) Relative levels of cellular YFP expression for the experiment shown in panel C, as determined by peak YFP output of cell lysates excited at 514 nm. (E) Gag-YFP(A206K)-Gag-CFP FRET curve for the membrane fraction (closed diamonds) and Gag-CFP curve for the membrane fraction (closed squares). Cytosolic fractions of Gag-YFP(A206K)-Gag-CFP are shown for comparison.