Figure 5.

Overexpression of Rab11 mutant proteins does not affect transport to late endosomal compartments. HeLa cells were transfected for 5 h with empty pGEM plasmid (control cells), Rab11Q70L, or Rab11S25N plasmids. (A) EGF (green fluorescence) and Tf (red fluorescence) were continuously internalized at 19°C for 45 min. In control cells and cells transfected with Rab11 mutants, EGF and Tf mostly colocalized in vesicular structures. (B) The cells were further chased at 37°C. After 5 min at 37°C, the staining for EGF (green fluorescence) appeared in vesicles separate from the vesicles containing Tf (red fluorescence) in control cells and Rab11Q70L cells. In Rab11S25N cells, internalized Tf reached the tubular network, as already seen in Fig. 3. Internalized EGF is located in vesicles separate from this network. (C) Measurement of ¹²⁵I-EGF degradation. After 5 h of transfection with empty pGEM plasmid (control cells, •), Rab11wt (♦), Rab11Q72L (▪), and Rab11S25N (□) plasmids, HeLa cells were incubated with ¹²⁵I-EGF on ice for 1 h. The cells were then washed in ice cold serum-free medium and incubated at 37°C for various times. Cell lysates and extracellular medium were collected and TCA precipitated. Extracellular TCA-soluble ¹²⁵I-EGF was calculated as a percentage of the total. No differences were observed in cells overexpressing Rab11wt and mutants compared with control cells.