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Fig. 7. Effects of ARL1 depletion on exocytosis and Golgi function. (A) VSG exocytosis assay in BSF line Bp2T7/ARL1 grown in the absence (No Tet) and presence (+Tet) of tetracycline for 24 hours. Parasites were labelled by pulse-chase using [35S]-labelled methionine and cysteine over a time course of 40 minutes and VSG isolated from cell lysates using ConA sepharose. Radiolabelled products were separated by SDS-PAGE and detected by autoradiography. Numbers represent length of pulse-chase (minutes). Insoluble pellet fractions (P) represent newly synthesized membrane-bound VSG in the endomembrane system, whereas soluble fractions (S) represent newly synthesized VSG delivered to the plasma membrane, where it is susceptible to the action of endogenous GPI-phospholipase C. The data shown represent one of three independent experiments. (B) Data from three assays as described in (A) were quantified by densitometry and mean values (±s.d.) are shown here as % VSG released on to the plasma membrane in BSF line Bp2T7/ARL1 grown in the absence (open squares) and presence (filled squares) of tetracycline for 24 hours. 0% is the ratio obtained at time 0 and 100% is the point at which all VSG is in the soluble fraction. (C,D) Immunoprecipitation of p67, following radioactive labelling of parasites by pulse chase with [35S]methionine and [35S]cysteine over a 4-hour time course. BSF line Bp2T7/ARL1 cells were grown in the absence (C) and presence (D) of tetracycline for 36 hours. Proteins were separated by SDS-PAGE and radiolabelled products detected by autoradiography. P67 is synthesized as a 100 kDa protein, which is modified by N-glycans to form a 150 kDa molecule, clearly visible in both samples by 30 minutes. After delivery to the lysosome, the protein is proteolytically cleaved into fragments of 75, 42, 32 and 28 kDa, as observed after 1 hour. Note that the lower fragment is not seen in this figure and that there is an unidentified band in samples from later time points with a molecular weight of approximately 55 kDa. (E,F) p67 immunoprecipitation results were quantified by densitometry in cells grown in the absence (light shading) and presence (dark shading) of tetracycline for 36 hours. (E) The percentage of total [35S]p67 present as the N-glycan modified glycoform (gp150) during the 4 hour pulse-chase time course. (F) Percentage of total [35S]p67 present as proteolytically cleaved fragments (gp75, 42 and 32 glycoforms) in the lysosome. Data shown represent one of two independent experiments.
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