Fig. 5. Effects of Rab22a on the uptake and degradation of rhodamine-conjugated epidermal growth factor (rho-EGF). Hep2 cells were infected with the Ankara strain Vaccinia T7 virus, followed by transfection of myc-tagged Rab22a wt, Q64L or S19N constructs in pGEM-1. Internalization (1 hour) and chase (3 hours) of rho-EGF were then carried out as described in Materials and Methods, and the cells were processed for confocal immunofluorescence microscopy. The rho-EGF fluorescence is shown in panels A,D,G,J,M,P, immunostaining for Rab22a in B,E,H,K,N,Q, and overlays in C,F,I,L,O,R. After the 1 hour internalization period, the rho-EGF was found in intracellular endocytic structures, the volume and distribution of which were highly similar in untransfected cells (arrows) and those expressing wt Rab22a (A-C), Rab22 Q64L (G-I) or Rab22 S19N (M-O). After 3 hours chase, the rho-EGF fluorescence had disappeared from untransfected cells and those expressing Rab22 S19N (P-R), although it remained detectable in cells expressing Rab22a wt (D-F) or the Q64L mutant (J-L). Colocalization of Rab22a wt (C,F) but not Q64L (I,L) was detected with the rho-EGF positive structures. Bar=10 µm.