Plant Molecular Biology 52: 259–271, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands. 259 Investigations on the in vitro import ability of mitochondrial precursor proteins synthesized in wheat germ transcription-translation extract Patrick Dessi 1 , Pavel F. Pavlov 1 , Fredrik Wållberg 1 , Charlotta Rudhe 1 , Simon Brack 1 , James Whelan 2 and Elzbieta Glaser 1,∗ 1 Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm Uni- versity, 10691 Stockholm, Sweden ( ∗ author for correspondence; e-mail e_glaser@dbb.su.se); 2 Department of Biochemistry, University of Western Australia, Nedlands, Australia Received 23 August 2002; accepted in revised form 17 October 2002 Key words: chimeric constructs, mitochondrial protein import, precursor, presequence, reticulocyte lysate, wheat germ extract Abstract Mitochondrial precursor proteins synthesized in rabbit reticulocyte lysate (RRL) are readily imported into mi- tochondria, whereas the same precursors synthesized in wheat germ extract (WGE) fail to be imported. We have investigated factors that render import incompetence from WGE. A precursor that does not require addi- tion of extramitochondrial ATP for import, the F A d ATP synthase subunit, is imported from WGE. Import of chimeric constructs between precursors of the F A d protein and alternative oxidase (AOX) with switched prese- quences revealed that the mature domain of the F A d precursor defines the import competence in WGE as only the construct containing the presequence of AOX and mature portion of F A d (pAOX-mF A d) could be imported. Import competence of F A d and pAOX-mF A d correlated with solubility of these precursors in WGE, however, solubilization of import-incompetent precursors with urea did not restore import competence. Addition of RRL to WGE-synthesized precursors did not stimulate import but addition of WGE to the RRL-synthesized precursors or to the over-expressed mitochondrial precursor derived from the F 1 β ATP synthase precursor inhibited import into mitochondria. The dual-targeted glutathione reductase precursor synthesized in WGE was imported into chloro- plasts, but not into mitochondria. Antibodies against the 14-3-3 guidance complex characterized for chloroplast targeting were able to immunoprecipitate all of the precursors tested except the F A d ATP synthase precursor. Our results point to the conclusion that the import incompetence of WGE-synthesized mitochondrial precursors is not presequence dependent and is a result of interaction of WGE inhibitory factors with the mature portion of precursor proteins. Abbreviations: AOX, alternative oxidase; cytochrome b 2 -DHFR, chimeric construct containing the presequence of yeast cytochrome b 2 and the mature portion of the cytosolic protein dihydrofolate reductase; F A d, Glycine max F A d subunit of ATP synthase; GR, glutathione reductase; MOPS, 3-(N -morpholino)propane sulfonic acid; MPB, 3-(N - maleimidopropionyl)biocytin; MPP, mitochondrial processing peptidase; MSF, mitochondrial import stimulation factor; mtHsp70, mitochondrial heat shock protein 70 kDa; MTX, methotrexate; N 15 pF 1 β 15 kDa N-terminal pF 1 β fragment; PK, proteinase K; PMSF, phenylmethylsulfonyl fluoride; RRL, Rabbit reticulocyte lysate; SDS- PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TIM, translocase of inner membrane; TOM, translocase of outer membrane; WGE, wheat germ extract