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. Author manuscript; available in PMC: 2013 Oct 19.
Published in final edited form as: J Biol Chem. 2002 Aug 20;277(43):40583–40593. doi: 10.1074/jbc.M203292200

Fig. 5. Interaction of phosphorylated CYP2E1 with mitochondrial protein translocases.

Fig. 5

A, 1–160 CYP2E1/DHFR, 1–160 S129A/DHFR, and 1–100 CYP1A1/DHFR fusion proteins translated in the RRL were used for in vitro import. B, effect of methotrexate-mediated translocation arrest of wild-type (1–160 CYP2E1/DHFR) and mutant (1-160 S129A/DHFR) fusion proteins. Reaction mixtures were preincubated on ice for 20 min with 1 µm methotrexate before initiating the import. C, chemical cross-linking of translocation-arrested 1–160 CYP2E1/DHFR and 1-160 S129A/DHFR fusion proteins with mitochondrial translocases and MtHsp70. Both fusion proteins were translated in presence of PKA (10 units), and translocation arrest was caused by adding 1 µm methotrexate as in B. IPP, immunoprecipitation; ab, antibody. Cross-linking with MBS cross-linker and immunoprecipitation were carried out as described under “Experimental Procedures.” In A and B, proteins were resolved by SDS-PAGE on 14% gels and in C on 10% gel. Cross-linked products (CLP) are indicated.