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Lysosome proteins are redistributed during expression of a GTP-hydrolysis-defective rab5a

Jennifer L. Rosenfeld1,4, Robert H. Moore2, K.-Peter Zimmer3, Estrella Alpizar-Foster4, Wenping Dai2, M. Nader Zarka1 and Brian J. Knoll4,*

1 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX 77030, USA
2 Department of Pediatrics, Pulmonary Section, Baylor College of Medicine, Houston, TX 77030, USA
3 Klinik und Poliklinik für Kinderheilkunde, Westfälische Wilhelms-Universität, Albert-Schweitzer-Str. 33, D-48149 Münster, Germany
4 Department of Pharmacological and Pharmaceutical Sciences, University of Houston, College of Pharmacy, Houston, TX 77204, USA



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  		Fig. 1. (A) Immunoblot analysis of EGFP-rab5aQ79L induction. An EcR293 cell line stably transfected with pIND-Hygro/EGFP-rab5aQ79L (EcR293 pIND-Hygro/EGFP-rab5aQ79L) was treated for 24, 48, 72 or 96 hours with 5 µM ponasterone or with vehicle (veh: 0.125% ethanol) for 96 hours only. Total cell lysates were electrophoresed through SDS-PAGE, blotted and probed with a monoclonal anti-rab5 antibody. Endogenous rab5 migrates at approximately 25 kDa, and EGFP-rab5aQ79L at 50 kDa. (B) Deconvolution fluorescence microscopy of EcR293/EGFP-rab5aQ79L cells treated for 72 hours with 5 µM ponasterone (pon) or vehicle (veh).

 


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  		Fig. 2. Rapid labeling of EGFP-rab5a-containing vesicles with transferrin Texas Red. EcR293 pIND-Hygro/EGFP-rab5a wt cells (A) and EcR293 pIND-Hygro/EGFP-rab5aQ79L cells (B) were grown for 72 hours in the presence of 5 µM ponasterone, then incubated for 5 minutes with transferrin Texas Red (20 µg/ml) prior to rapid fixation in 4% paraformaldehyde. The cells were imaged by deconvolution fluorescence microscopy. Bar, 10 µm.

 


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  		Fig. 3. Lysosome/late endosome proteins localize with EGFP-rab5aQ79L. EcR293 pIND-Hygro/EGFP-rab5aQ79L cells were treated with vehicle (left) or 5 µM ponasterone (right) for 72 hours and then fixed and labeled with antibodies against LAMP-1, LAMP-2, cathepsin D or CI-M6PR as indicated. Secondary antibodies were TRITC (red) goat anti-mouse or goat anti-rabbit IgG. The labeled cells were imaged by deconvolution fluorescence microscopy. Bar, 10 µm.

 


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  		Fig. 4. Immunogold labeling and electron microscopy of EGFP-rab5aQ79L-expressing cells. EcR293pIND-Hygro /EGFP-rab5aQ79L cells were grown in the presence of ponasterone for 72 hours, then fixed in 4% PFA and processed for immunogold labeling and electron microscopy as described in Materials and Methods. EGFP, 6 nm gold (arrowheads); LAMP-2, 12 nm gold (arrows); M, mitochondrion. Magnification, 65,000x.

 


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  		Fig. 5. LAMP-1 distribution in cells expressing EGFP-rab5a wt or EGFP-rab5aS34N. EcR293pIND-Hygro /EGFP-rab5a wt cells (A) and EcR293pIND-Hygro/EGFP-rab5a S34N cells (B) were induced for 72 hours with 5 µM ponasterone, then fixed and labeled with anti-LAMP-1 primary and TRITC (red) secondary antibodies. The labeled cells were imaged by deconvolution fluorescence microscopy. Bar, 10 µm.

 


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  		Fig. 6. Endocytic markers accumulate in EGFP-rab5aQ79L-containing vesicles. (A,B). EcR293pIND-Hygro /EGFP-rab5a wt cells (A) and EcR293pIND-Hygro/EGFP-rab5aQ79L cells (B) were induced with 5 µM ponasterone for 72 hours, then pulse labeled for 1 hour with dextran Texas Red, followed by a 6 hour chase prior to fixation. (C,D). EcR293 pIND-Hygro/EGFP-rab5a wt cells (C) and EcR293 pIND-Hygro/EGFP-rab5aQ79L cells (D) were induced with 5 µM ponasterone for 72 hours, then pulse labeled for 5 minutes with Di-LDL, followed by a 1 hour chase prior to fixation. The labeled cells were imaged by deconvolution fluorescence microscopy. Bar, 10 µm.

 


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  		Fig. 7. Endocytosis and recycling of a cell surface receptor. EcR293 pIND-Hygro/EGFP-rab5aQ79L cells were stably transfected with pcDNA3.1/ß2AR, then incubated with either vehicle ({blacksquare}) or 5 µM ponasterone ({square}) for 72 hours. The cells were treated with 10 µM isoproterenol for varying times, then chilled, washed and kept on ice with 6 nM [3H]CGP12177 for 90 minutes to selectively label surface receptors. The fraction of receptors left on the surface was plotted versus time of agonist exposure, and the curves modeled as described (Morrison et al., 1996) to obtain estimates for the first-order rate constants of endocytosis (ke) and recycling (kr). The rate constant ke for control cells was 0.199±0.010 minutes–1 and for ponasterone-treated cells, 0.151±0.005 minutes–1 (significantly different, P=0.029). The rate constant kr was 0.053±0.005 minutes–1, and for ponasterone-treated cells, 0.038±0.010 minutes–1 (not significant, P=0.1005) (n=3).

 


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  		Fig. 8. Dextran Texas Red preloaded into lysosomes localizes with EGFP-rab5aQ79L. Cells were incubated with dextran Texas Red (1 mg/ml) for 1 hour, washed and then chased for 6 hours in the absence of tracer. The cells were then induced with 5 µM ponasterone or vehicle for 72 hours prior to fixation, and imaged by deconvolution fluorescence microscopy. (A) EcR293 pIND-Hygro/EGFP-rab5a wt; (B) EcR293 pIND-Hygro/EGFP-rab5aQ79L; (C) EcR293 pIND-Hygro/EGFP-rab5aQ79L pulse labeled with dextran Texas Red and incubated 72 hours with vehicle alone. Bar, 10 µm.

 


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  		Fig. 9. Distribution of ß-hexosaminidase in Percoll gradients. EcR293 pIND-Hygro/EGFP-rab5aQ79L cells were treated with vehicle ({blacksquare}) or ponasterone ({square}) for 72 hours, then homogenized and applied to self-forming Percoll gradients (see Materials and Methods). After centrifugation, samples were taken from the bottom (fraction 1), and aliquots assayed for ß-hexosaminidase activity and protein concentration. Results are representative of three independent experiments.

 

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