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Peroxisomal membrane proteins are properly targeted to peroxisomes in the absence of COPI- and COPII-mediated vesicular transport

Tineke Voorn-Brouwer, Astrid Kragt, Henk F. Tabak and Ben Distel*

Department of Biochemistry, University of Amsterdam, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands



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  		Fig. 1. PMPs are rapidly targeted to peroxisomes. Normal human fibroblasts were microinjected with DNA constructs expressing Pex2p-GFP (A,B), Pex3p-GFP (C,D), Pex16p-GFP (E,F) and GFP-PMP70 (G,H). After 90 minutes, cells were fixed and processed for double indirect immunoflourescence with antibodies to GFP (A,C,E,G) and ALDP (B,D,F,H).

 


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  		Fig. 2. Peroxisomal targeting of PMPs is not inhibited by BFA. Normal human fibroblasts were incubated in either the presence (A-J) or the absence (K,L) of 2 µg ml-1 BFA for 20 minutes and then microinjected with DNA constructs expressing Pex2p-GFP (A,B), Pex3p-GFP (C,D), Pex16p-GFP (E,F), GFP-PMP70 (G,H) and HA (I-L). After microinjection, the cells were maintained in mock or BFA-containing media for 90 minutes and then processed for double indirect immunofluorescence with antibodies to GFP (A,C,E,G) and ALDP (B,D,F,H), or with antibodies to HA (I,K) and PDI (J,L).

 


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  		Fig. 3. Inhibitors of COPI and COPII do not affect peroxisome morphology. Normal human fibroblasts were incubated either in the absence (A,B) or in the presence (C,D) of 2 µg ml-1 BFA. After 20 hours, cells were fixed and stained with antibodies to acyl-CoA-oxidase (A,C) or giantin (B,D). NH-tagged Sar1p(H79G) DNA was microinjected into the nucleus of normal human fibroblasts. After 20 hours, cells were processed for double indirect immunofluorescence with antibodies to catalase (E) and the NH epitope (F), or with antibodies to ERGIC53 (G) and the NH epitope (H).

 


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  		Fig. 4. Inhibition of COPII-mediated trafficking does not affect PMP targeting to peroxisomes. Normal human fibroblasts were co-injected with either NH Sar1p(H79G) and constructs expressing PMP-GFP fusions (Pex2p-GFP (A,B), Pex3p-GFP (C,D), Pex16p-GFP (E,F), GFP-PMP70 (G,H)) or NH Sar1p(H79G) and HA (I,J). After 5 hours, cells were fixed and processed for double indirect immunofluorescence with antibodies to GFP (A,C,E,G) and ALDP (B,D,F,H), or with antibodies to HA (I) and PDI (J).

 

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© The Company of Biologists Ltd 2001