Investigations on the in vitro import ability of mitochondrial precursor proteins synthesized in wheat germ transcription-translation extract.
Mitochondrial precursor proteins synthesized in rabbit reticulocyte lysate (RRL) are readily imported into mitochondria, 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 addition 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 presequences 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 F1beta ATP synthase precursor inhibited import into mitochondria. The dual-targeted glutathione reductase precursor synthesized in WGE was imported into chloroplasts, 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.
Dessi
Patrick
P
Department of Biochemistry and Biophysics, Arrhenius Laboratories for Natural Sciences, Stockholm University, 10691 Stockholm, Sweden.
Pavlov
Pavel F
PF
Wållberg
Fredrik
F
Rudhe
Charlotta
C
Brack
Simon
S
Whelan
James
J
Glaser
Elzbieta
E
D048948:14-3-3 Proteins; D000818:Animals; D002474:Cell-Free System / Q000378:metabolism; D002736:Chloroplasts / Q000187:drug effects / Q000378:metabolism; D005980:Glutathione Reductase / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics* / Q000378:metabolism; D010936:Plant Extracts / Q000235:genetics / Q000494:pharmacology; D011233:Precipitin Tests; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics*; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D012995:Solubility; D014158:Transcription, Genetic / Q000235:genetics*; D014908:Triticum / Q000235:genetics; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism
D048948:14-3-3 Proteins; D018840:HSP70 Heat-Shock Proteins; D024101:Mitochondrial Proteins; D010936:Plant Extracts; D011498:Protein Precursors; D014446:Tyrosine 3-Monooxygenase; D005980:Glutathione Reductase