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First published online 18 January 2005
doi: 10.1242/jcs.01645

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Direct transport across the plasma membrane of mammalian cells of Leishmania HASPB as revealed by a CHO export mutant

¹ Heidelberg University Biochemistry Center, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany
² Zentrum für Molekulare Biologie Heidelberg, 69120 Heidelberg, Germany
³ Wellcome Trust Laboratories for Molecular Parasitology, Department of Biological Sciences, Imperial College London, SW7 2AZ, UK

View larger version (32K): [in a new window]   Fig. 1. Expression level and membrane association of HASPB-GFP fusion proteins expressed in CHO cells. (A) CHO cells expressing HASPB-GFP fusion proteins as indicated were grown on six-well plates in the absence (lanes 1, 3, 5 and 7) or presence (lanes 2, 4, 6 and 8) of 1 µg/ml doxicycline (dox). Cells were detached and collected by centrifugation followed by lysis in SDS sample buffer. 1% of each lysate corresponding to cells from one well were subjected to SDS-PAGE. Following SDS-PAGE and western blotting HASPB-GFP fusion proteins were detected using affinity-purified anti-GFP antibodies. (B) CHO cells expressing HASPB-GFP fusion proteins as indicated were fractionated into cytosolic (lane 1) and membrane fractions (lane 2). Additionally, the membrane fraction was subjected to carbonate extraction resulting in a carbonate supernatant containing loosely bound proteins (lane 3) and a carbonate pellet containing proteins tightly associated with membranes (lane 4). 5% of each fraction was combined with SDS sample buffer and proteins were separated by SDS-PAGE. Following western blotting, HASPB-GFP fusion proteins were detected with affinity-purified anti-GFP antibodies.

View larger version (167K): [in a new window]   Fig. 2. Subcellular distribution of HASPB-GFP fusion proteins as determined by confocal microscopy. Cells were grown on glass coverslips in the presence of 1 µg/ml doxicycline for 48 hours at 37°C. GFP-derived fluorescence was viewed with a Zeiss LSM 510 confocal microscope. (A) HASPB-N18-GFP; (B) HASPB-N18-GFP-palm; (C) HASPB-N18-GFP-myr/palm; (D) GFP.

View larger version (43K): [in a new window]   Fig. 3. Quantitative analysis of cell surface localized HASPB-GFP fusion proteins using flow cytometry. (A) GFP-derived fluorescence (expression level). (B) APC-derived fluorescence (cell surface staining). Cells were incubated in the presence or absence of doxicycline as indicated followed by processing for FACS sorting. HASPB-N18-GFP, light and dark blue curves; HASPB-N18-GFP-palm, yellow and orange curves; HASPB-N18-GFP-myr/palm, light and dark green curves; GFP, light and dark red curves.

View larger version (28K): [in a new window]   Fig. 4. Biochemical quantification of cell surface localized HASPB-GFP fusion proteins. CHO cells expressing HASPB-GFP fusion proteins as indicated were treated with a membrane-impermeable biotinylation reagent. Cell lysates were generated and biotin-labeled and biotin-unlabeled proteins were separated by streptavidin affinity chromatography. Input material (lane 1; 2%), streptavidin supernatant (non-biotinylated proteins, lane 2; 2%) and streptavidin-bound proteins (biotinylated proteins, lane 3; 50%) were separated on SDS gels followed by western blotting using affinity-purified anti-GFP antibodies.

View larger version (36K): [in a new window]   Fig. 5. Retroviral insertion mutagenesis of CHO cells and genetic screening for HASPB export mutants. (A) Intercellular spreading was monitored by growing CHOMCAT-TAM2 cells ('CHO wild type') with CHOMCAT-TAM2 cells retrovirally transduced with the HASPB-N18-GFP construct in a mixed culture. HASPB-N18-GFP expression was induced by 1 µg/ml doxicycline for 48 hours at 37°C. Cells were then processed for FACS sorting using affinity-purified anti-GFP antibodies and APC-coupled secondary antibodies to detect exported HASPB-N18-GFP by cell surface staining. CHOMCAT-TAM2 cells and CHOMCAT-TAM2 cells expressing HASPB-N18-GFP were gated based on GFP fluorescence and APC-derived fluorescence of CHOMCAT-TAM2 cells (green curve) and CHOMCAT-TAM2 cells expressing HASPB-N18-GFP (blue curve) was measured as depicted in the histogram. Autofluorescence was measured using CHOMCAT-TAM2 cells that were treated with antibodies. (B) CHOMCAT-TAM2 cells expressing HASPB-N18-GFP were treated with retroviral particles encoding the cell surface protein CD4. Following transduction, CD4-positive cells were selected based on anti-CD4 cell surface staining using FACS sorting. (C) CD4-positive cells as enriched in the FACS experiment depicted in B were subjected to three rounds of FACS sorting. Cells were monitored in dot blot mode with GFP-derived fluorescence shown on the y-axis and HASPB-N18-GFP cell surface staining shown on the x-axis. The left-hand panel shows the population grown in the absence of doxicycline, the right-hand panel shows the population grown in the presence of 1 µg/ml doxicycline for 48 hours at 37°C. To select for HASPB export mutants the sorting window was adjusted as depicted in the right-hand panel to isolate cells characterized by high GFP fluorescence and low APC cell surface staining.

View larger version (33K): [in a new window]   Fig. 6. Characterization of HASPB-N18-GFP export from CHO wild-type cells compared to CHO K3 mutant cells. (A) FACS analysis. CHO wild-type cells and CHO K3 cells were grown for 48 hours at 37°C in the presence of doxicycline (1 µg/ml). Cells were processed for FACS sorting using affinity-purified anti-GFP antibodies and APC-coupled secondary antibodies to detect exported HASPB-N18-GFP by cell surface staining. For a statistical analysis of four independent experiments, GFP-derived fluorescence and APC-derived cell surface staining of CHO wild-type cells expressing HASPB-N18-GFP was set to 100%, respectively. (B) Biochemical analysis of exported HASPB-N18-GFP in CHO wild-type cells, CHO K3 cells and various control cell lines introduced in Figs 1, 2, 3, 4 using cell surface biotinylation. The experiment was conducted exactly as described in the Materials and Methods and in the legend to Fig. 4. Input material (lane 1; 2%), streptavidin supernatant (non-biotinylated proteins, lane 2; 2%) and streptavidin-bound proteins (biotinylated proteins, lane 3; 50%) were separated on SDS gels followed by western blotting using affinity-purified anti-GFP antibodies.

View larger version (26K): [in a new window]   Fig. 7. Expression level, membrane association and post-translational acylation of HASPB-N18-GFP in CHO wild-type cells and CHO mutant K3 cells. (A) CHO wild-type cells and CHO K3 cells (both expressing HASPB-N18-GFP) were grown on six-well plates to about 80% confluency in the absence (lane 1) or presence (lane 2) of doxicycline (1 µg/ml) for 48 hours at 37°C. Cells were detached with PBS/EDTA, collected by centrifugation and lysed in SDS sample buffer. 1% of each lysate corresponding to cells from one well were subjected to SDS-PAGE. HASPB-N18-GFP was detected by western blotting using affinity-purified anti-GFP antibodies. (B) CHO wild-type cells and CHO K3 cells (both expressing HASPB-N18-GFP) were grown on six-well plates to about 80% confluency in the presence of 1 µg/ml doxicycline for 48 hours at 37°C. Subcellular fractionation and carbonate extraction of membranes was performed and 5% of each fraction was combined with SDS sample buffer and proteins were separated by SDS-PAGE. Following western blotting, HASPB-GFP fusion proteins were detected with affinity-purified anti-GFP antibodies. (C) CHO wild-type cells, CHO K3 cells (both expressing HASPB-N18-GFP) as well as control cell lines expressing HASPB-N18-GFP myr/palm and HASPB-N18-GFP palm, respectively, were grown on six-well plates to about 80% confluency in the presence of 1 µg/ml doxicycline for 48 hours at 37°C and labelled with [3H]myristate and [3H]palmitate. Cell lysates were prepared and subjected to immunoprecipitation using affinity-purified anti-GFP antibodies. Immunopurified fractions were split into two samples, separated on SDS gels and either processed by fluorography (upper panel) or silver staining (lower panel).

View larger version (53K): [in a new window]   Fig. 8. Subcellular localization of HASPB-N18-GFP in CHO wild-type and CHO K3 mutant cells as determined by confocal microscopy and subcellular fractionation. (A) HASPB-N18-GFP expressed in CHO wild-type cells. (B) HASPB-N18-GFP expressed in CHO K3 mutant cells. Cells were grown on glass coverslips in the presence of 1 µg/ml doxicycline for 48 hours at 37°C and processed for confocal microscopy. GFP-derived fluorescence was viewed with a Zeiss LSM 510 confocal microscope. (C) Subcellular fractionation of CHO wild-type cells and CHO K3 cells was conducted as described earlier (Schäfer et al., 2004). To identify plasma membranes, antibodies directed against the transferrin receptor were used (Futter et al., 1998). To detect Golgi membranes, antibodies directed against GM130 were used (Nakamura et al., 1995). Four fractions were generated and analyzed for each cell line: a hypotonic lysate (lanes 1 and 5), a post-mitochondrial supernatant (lanes 2 and 6), a microsomal membrane fraction (lanes 3 and 7) and gradient-purified plasma membranes (lanes 4 and 8). For each fraction 15 µg total protein were loaded per lane followed by SDS-PAGE and western blotting using the antibodies indicated.

View larger version (43K): [in a new window]   Fig. 9. Secretion of FGF-2 from CHO K3 cells occurs as efficiently as from parental CHO wild-type cells. Parental CHO wild-type (lanes 1-3) and CHO K3 mutant cells (lanes 4-6) expressing HASPB-N18-GFP were transduced with retroviral particles containing the FGF-2-GFP open reading frame controlled by a doxicycline-dependent element. Transduction efficiency was about 65% as determined by GFP-derived fluorescence. Both cell types were treated with a membrane-impermeable biotinylation reagent. Cell lysates were generated and biotin-labeled and biotin-unlabeled proteins were separated by streptavidin affinity chromatography. Input material (lane 1 and 4; 4%), streptavidin supernatant (non-biotinylated proteins, lanes 2 and 5; 4%) and streptavidin-bound proteins (biotinylated proteins, lanes 3 and 6; 50%) were separated on SDS gels followed by western blotting using affinity-purified anti-GFP antibodies.

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