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ABC transporters required for endocytosis and endosomal pH regulation in Dictyostelium

Derrick T. Brazill^1,*, Lowell R. Meyer², R. Diane Hatton¹, Debra A. Brock¹ and Richard H. Gomer^1,2,

¹ Howard Hughes Medical Institute
² Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892, USA
^* Present address: Department of Biological Sciences Hunter College, Rm 927 North Building, 695 Park Avenue, New York, NY 10021, USA

View larger version (18K): [in a new window]   Fig. 1. Disruption of mdrA1 and mdrA2 results in a partial rescue of the developmental defect exhibited by mutation of rtoA. Wild-type DH1 cells, rtoA- cells, rtoA-/mdrA1-/mdrA2- cells and mdrA1-/mdrA2- cells were plated with bacteria on agar. As the bacteria were consumed, development was triggered and after 24 hours fruiting structures formed. A side view of these resulting developmental structures is shown. The width of each frame is 3.5 mm.

View larger version (69K): [in a new window]   Fig. 2. MdrA1 and MdrA2 have sequence similarity to known ABC transporter proteins. The complete nucleotide and derived amino acid sequence of mdrA1 and mdrA2 are available in GenBank as AF246689. The N-terminal and C-terminal ABC transporter domains of MdrA1 and MdrA2 are aligned with the consensus ABC transporter domain from the Pfam protein families database (Bateman et al., 2000). The alignment was done using the program CLUSTAL W (Thompson et al., 1994). Black boxes indicate identical amino acids whereas shaded boxes indicate conservatively substituted residues.

View larger version (44K): [in a new window]   Fig. 3. The expression of mdrA2 is developmentally regulated. A northern blot of total cellular RNA isolated from DH1 cells at 2.5 hour intervals after starvation on filter pads was probed with a radiolabelled fragment of the mdrA2 gene (A). A western blot of lysates from 1x105 cells of mdrA1-/mdrA2-, DH1, rtoA- and rtoA-/mdrA1-/mdrA2- was probed with affinity-purified anti-MdrA2 peptide antibodies (B). A similar western was performed using 1x105 cells of DH1 at 2.5 hour intervals after starvation on filter pads (C). mdrA–, mdrA1-/mdrA2-; rtoA–/mdrA–, rtoA-/mdrA1-/mdrA2-.

View larger version (32K): [in a new window]   Fig. 4. Differentiation of rtoA-/mdrA1-/mdrA2- cells. Cells were grown in low-density monolayer culture overnight, with a field of the cells being videotaped. The medium was then gently changed to conditioned starvation buffer, and 6 hours later cAMP was added to induce the expression of cell-type-specific markers. Eighteen hours after starvation the cells were fixed, the videotape was turned off, and the cells were stained for the prestalk marker CP2 and the prespore marker SP70. The time of cytokinesis and the fate of the sister cell for each positive cell were then determined by examining the videotape. Open circles mark the phase that SP70-positive prespore cells happened to be in at the time of starvation, whereas filled squares mark where CP2-positive prestalk cells happened to be.

View larger version (59K): [in a new window]   Fig. 5. Immunolocalization of MdrA1. Wild-type DH1 (A-D) and rtoA- (E-H) cells were fixed and stained with anti-MdrA1 antibody raised against the GST-MdrA1 fusion protein (B, D, F, H). The corresponding phase images are shown in A, C, E and G. The cells were either taken directly from growth media (A, B, E, F) or osmotically stressed (C, D, G, H) before fixation. Bar, 10 µm.

View larger version (19K): [in a new window]   Fig. 6. (A) Endocytosis rates in wild-type, mdrA1-/mdrA2- and rtoA-/mdrA1-/mdrA2- cells. Cells were collected and resuspended in HL5 containing FITC-dextran. At the times indicated by the symbols, cells were removed, washed twice in HL5 and once with wash buffer. They were then lysed in wash buffer containing Triton X-100 and the fluorescence measured as described in Materials and Methods. (B) Exocytosis rates in wild-type, mdrA1-/mdrA2- and rtoA-/mdrA1-/mdrA2- cells. Cells were allowed to internalize FITC dextran for 3 hours; they were then washed and resuspended in fresh HL5. At the times indicated by the symbols, cells were collected by centrifugation, washed once and lysed in wash buffer containing Triton X-100. The fluorescence was then measured as discussed in Materials and Methods. Values are mean±s.e.m. mdrA-, mdrA1-/mdrA2-; rtoA-/mdrA, rtoA-/mdrA1-/mdrA2-.

View larger version (19K): [in a new window]   Fig. 7. Endosomal pH in wild-type, rtoA-, mdrA1-/mdrA2- and rtoA-/mdrA1-/mdrA2- cells. Cells were fed FITC-dextran for 10 minutes, collected, and resuspended in buffer. At the indicated times, aliquots of cells were removed, centrifuged, and resuspended in assay buffer, and fluorescence was measured using excitation at 450 and 495 nm. The pH values were obtained from a standard curve of the FITC dextran fluorescence ratio as a function of pH. Values are mean±s.e.m.

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