Figure 6: Covalently locked Rab5 does not localize to endosomes.

(a) GFP–Rab5_WT non-covalently bound to GDP (left) or GppNHp (middle) and GFP–Rab5_Q79L non-covalently bound to GTP (right) were microinjected into cells. All constructs show a localization resembling that expected for early endosomes. While Rab5_Q79L induces formation of enlarged endosomes as previously reported³⁴, this is not the case for Rab5_WT independently of the nucleotide-bound state (that is, GDP or GppNHp) before microinjection. (b) GFP–Rab5_E47C–aGDP microinjected into cells shows strong localization to the Golgi apparatus instead of endosomes as indicated by co-staining with Giantin and Cherry-Rab5_WT (Cherry-Rab5_WT was used as a marker of correct intracellular targeting of Rab5 to endosomes). (c) GFP–Rab5_{E47C, Q79L}–aGTP microinjected into cells is localized to the Golgi apparatus, similar to Rab5–aGDP. This is presumably due to the low remaining hydrolysis activity of Rab5_Q79L, resulting in the inactive aGDP-bound conformation within the time frame of the experiment. (d) GFP–Rab5_E47C–aGppNHp microinjected into the cell shows a distribution over endomembranous structures throughout the cell with a preference for the endoplasmic reticulum (upper panel; cb5, cytochrome b5), but not specifically to endosomes (lower panel). (e) Cells transfected with eGFP-Rab5_E47C show a similar localization pattern of Rab5_E47C compared with Rab5_WT. This clearly shows that the E47C mutation alone does not affect intracellular localization (scale bars: 10 μm).