In the course of this study Pex7p, Pex14p and Pex8p were identified as new interaction partners of Pex8p, by means of two-hybrid-analysis. Interactions of further peroxines of the docking-machinery, to peroxines of the zincfinger-class and of other functional groups were not identified. The region of interaction of Pex8p and Pex5p, Pex8p and Pex14p was narrowed down to amino acids 146-586 of Pex8p. It was not possible to narrow down the region of interaction of Pex8p to Pex7p. Pex7p needs a full length Pex8-protein for interaction. For interaction with Pex8p (146-586 aa) Pex5p requires the amino acids 1-312. The TRP-domains of Pex5p are not required for the interaction between Pex5p and Pex8p. The interaction between Pex8p and Pex14p is influenced by peroxines of the "cargo-receptor-binding-group", the "docking-machinery-group", and the "receptor-translocation-group", and is therefore indirect. The interactions between Pex8p and Pex5p, as well as between Pex8p and Pex7p are not mediated by peroxines of the investigated functional groups and are therefore likely to be direct. By means of mutationanalysis of Pex8p, three mutations were identified, which prevent interactions between Pex8p and Pex5p, Pex7p and Pex14p in different ways. Mutation Pex8pM-A(S231P) prevents the interaction to Pex5p, Pex7p and Pex14p. Mutation Pex8pM-C(A238V) prevents the interaction to Pex5p and Pex14p. Mutation Pex8pM-D(T327P) prevents the interaction to Pex7p and Pex14p. Therefore, both receptors seem to be necessary for interaction between Pex8p and Pex14p. The Pex8p-mutant can be complemented by all mutated Pex8-proteins. Moreover, fluorescently labelled cargoproteins are imported into peroxisomes in complemented pex8D-cells. On the basis of the mutationanalysis of Pex8p it is likely, that Pex8p induces the release of the cargo-receptorcomplex due to changes of the receptor conformation. In spite of the presence of a PTS-signal in Pex8p, the targeting of Pex8p to the peroxisomal membrane is independent of the PTS-receptors. This is further supported by extraction analysis of the protein from peroxisomal membranes. In the absence of Pex5p, Pex8p can be degraded by Proteinase K. Therefore Pex5p does not influence the targeting of Pex8p but indirectly influences its approachability to the cytosolic components.
570 Life Sciences ; Ddc:570 ; 570 Biowissenschaften; Biologie ; Saccharomyces ; Peroxisome ; Protein ; Import ; Pex8
Freie Universitat Berlin
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