Figure 5.

Relocated lysosomes maintain some structural and functional identities. (A) NRK cells were transfected with Rab34Q111L and then processed to detect the Golgi apparatus marked by syntaxin 6 (a) and lysosomes marked by Lamp1 (b). As shown, redistributed lysosomes seem to embrace the Golgi apparatus. Bar, 10 μm. (B) NRK cells transfected with EGFP-Rab34Q111L was treated with brefeldin A (a–c) or nocodazole (d–f) (both at 10 μg/ml for 60 min at 37°C) and then processed to detect lysosomes by anti-Lamp1 labeling. As shown, lysosomal repositioning induced by EGFP-Rab34Q111L was not affected by brefeldin A but was randomized by nocodazole treatment. Bar, 10 μm. (C) Control or Hela cells transfected using Effectence (efficiency of transfection was estimated to be ∼60–70% by viewing EGPF positive cells) with constructs for expressing indicated proteins were processed for immunoblot analysis to detect various forms of cathepsin D (P for Golgi-associated form, I for endosomal intermediate, and M for mature lysosomal form) (top). As shown, the appearance of mature lysosomal form of cathepsin D was not affected by expression of any form of Rab34, suggesting that transport of cathepsin D from the Golgi to the lysosome was not significantly affected. The expressed EGFP and EGFP-Rab34 were revealed to be present at comparable levels in these transfected cells (middle), whereas the loading was normalized to the amount of ribophorin (bottom).