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First published online 18 January 2005
doi: 10.1242/jcs.01627

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Dissection of brefeldin A-sensitive and -insensitive steps in apicoplast protein targeting

¹ Seattle Biomedical Research Institute, 307 Westlake Avenue N., Suite 500 Seattle, WA 98109, USA
² Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, WA 98195, USA

View larger version (48K): [in a new window]   Fig. 1. An ER retrieval signal does not block targeting to the apicoplast. (A) T. gondii expressing ACP-GFP-HDEL and S9(S)-GFP-HDEL were analyzed by IFA and visualized with anti-GFP, and anti-BiP [S9(S)-GFP-HDEL only], the apicoplast marker quantum red streptavidin and DAPI. A merge of anti GFP or anti BiP, quantum red streptavidin and DAPI is also shown. A merged projection of the marked apicoplasts in the y-z dimension is shown. (B) Anti-GFP immunoblot analysis of protein from ACP-GFP-HDEL (lane 1) and ACP-GFP (lane 2) parasites. t-GFP indicates the species containing the transit peptide and m-GFP the mature form lacking the transit peptide. Bar, 5 µm.

View larger version (26K): [in a new window]   Fig. 2. Kinetics of cleavage of the apicoplast transit peptide. Intracellular T. gondii expressing ACP-GFP were labeled with 35S amino acids for 1 hour, and chased for 0, 1, 2 and 4.75 hours in control medium. GFP was immunoprecipitated, proteins resolved by SDS-PAGE, and visualized with a phosphorimager. Transit (t-GFP) and mature (m-GFP) forms of ACP-GFP were detected.

View larger version (47K): [in a new window]   Fig. 3. BFA blocks transit peptide cleavage. (A) Intracellular T. gondii expressing ACP-GFP were labeled with 35S amino acids for 30 minutes (pulse) then chased for 4 hours in medium containing the indicated concentrations (ng/ml) of BFA. GFP and Mic5 were immunoprecipitated, resolved by SDS-PAGE and detected by phosphorimaging. Transit peptide (t-GFP) and mature forms (m-GFP) of GFP as well as intermediate (i-mic5) and mature forms (m-mic5) of Mic5 are indicated. (B) Quantification of GFP (diamonds) and Mic5 (squares) processing calculated based on the maximum processing measured in the experiment shown in A.

View larger version (32K): [in a new window]   Fig. 4. The BFA-sensitive step in apicoplast protein import occurs before transit peptide cleavage. Intracellular T. gondii expressing ACP-GFP were labeled with 35S amino acids in the presence of 1 µg/ml BFA for 1 hour (pulse) then chased in control medium for various times (-BFA, hours). Alternatively (three right lanes), the samples were chased without BFA for the indicated times (-BFA, hours) and then BFA was added to 1 µg/ml and the incubation continued for an additional period (+BFA, hours). In these BFA add-back experiments, the total incubation time was 4 hours. GFP was immunoprecipitated and resolved by SDS-PAGE; t-GFP marks the species containing the transit peptide, m-GFP marks the mature form in which lacks the transit peptide. The amount of precursor and mature protein was quantified by phosphorimaging and the percentage of protein in the mature form is indicated below each lane.

View larger version (53K): [in a new window]   Fig. 5. Extended treatment with BFA does not impede GFP localization to the apicoplast. T. gondii expressing ACP-GFP, or ACP-GFP-HDEL were grown in control medium (con.) with or without 1 µg/ml BFA for 4 hours. Shown are IFAs probed with anti-GFP and the apicoplast marker quantum red streptavidin. A merge of anti-GFP, quantum red streptavidin and DAPI is shown. Bar, 5 µm.

View larger version (43K): [in a new window]   Fig. 6. The conditional aggregation domain system can modulate protein trafficking from the ER. (A) T. gondii stably transfected with S9(S+T)-CAD-GFP were grown overnight in the absence of ligand, and then ligand was added for the indicated times before fixation. Shown are IFAs probed with anti-GFP and quantum red streptavidin. A merge of anti-GFP, quantum red streptavidin and DAPI is shown. (B) GFP fluorescence of S9(S)-CAD-GFP transfected T. gondii grown without ligand (-) and overnight with ligand (+). Arrow indicates parasitophorous vacuole. Lines were drawn on the same images to assist in identification of the parasites and the parasitophorous vacuole membrane (lower panels). (C) Protein was extracted from S9(1-159)-CAD-GFP parasites that had been treated with ligand for 40 minutes (lane 1), grown overnight without ligand (lane 2), and grown overnight in ligand (lane 3). Samples were separated by SDS-PAGE, transferred to nitrocellulose, and probed with anti-GFP and anti-NTPase antibodies. Bar, 5 µm.

View larger version (67K): [in a new window]   Fig. 7. BFA does not impede ligand-mediated localization of S9(S+T)-CAD-GFP to the apicoplast. (A) Effect of BFA on apicoplast targeting. T. gondii stably transfected with S9(S+T)-CAD-GFP were grown for 3 days in the absence of ligand. Samples were incubated in one of several conditions prior to fixation as indicated: BFA, no ligand for 1 hour; BFA for 5 minutes followed by ligand for 20 minutes or 240 minutes; ligand alone for 20 minutes or 240 minutes. Samples were prepared for IFA, anti-GFP and quantum red streptavidin are shown, and a merge of anti-GFP, quantum red streptavidin and DAPI. (B) Effect of BFA on secretion. T. gondii stably transfected with S9(S)-CAD-GFP were grown overnight in the absence of ligand. GFP fluorescence in live cells was viewed at time 0 and then in the same cells 30 minutes after the addition of ligand or ligand plus BFA. As with apicoplast proteins, BFA was added 5 minutes before ligand. Arrow indicates parasitophorous vacuole. Lines were drawn on the same images to facilitate the localization of parasites and the parasitophorous vacuole membrane (lower panels). Bar, 5 µm.

View larger version (61K): [in a new window]   Fig. 8. Ligand-mediated relocalization of S9(S+T)-CAD-GFP to the apicoplast occurs at low temperature. (A) Effect of low temperature on apicoplast targeting. T. gondii stably transfected with S9(S+T)-CAD-GFP were grown for 3 days without ligand, and transferred to a water bath equilibrated at 15°C. After 15 minutes, pre-chilled medium containing ligand was added and the cells incubated for the indicated times. Samples were prepared for IFA and probed with anti-GFP and quantum red streptavidin. A merge of GFP, quantum red streptavidin and DAPI is shown. (B) Effect of low temperature on secretion. T. gondii stably transfected with S9(S)-CAD-GFP were grown overnight in the absence of ligand and transferred to a water bath equilibrated at 15°C. After 15 minutes, ligand was added as above and the cultures were incubated for 3.5 hours before mild fixation. Cells were viewed for GFP fluorescence and transmitted light. Arrows indicate parasitophorous vacuole. Cartoons are overlaid on the images to facilitate identification of the parasites and the parasitophorous vacuolar membrane. Bar, 5 µm.

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