Isolated plant mitochondria import chloroplast precursor proteins in vitro with the same efficiency as chloroplasts.
Most chloroplast and mitochondrial proteins are synthesized with N-terminal presequences that direct their import into the appropriate organelle. In this report we have analyzed the specificity of standard in vitro assays for import into isolated pea chloroplasts and mitochondria. We find that chloroplast protein import is highly specific because mitochondrial proteins are not imported to any detectable levels. Surprisingly, however, pea mitochondria import a range of chloroplast protein precursors with the same efficiency as chloroplasts, including those of plastocyanin, the 33-kDa photosystem II protein, Hcf136, and coproporphyrinogen III oxidase. These import reactions are dependent on the Deltaphi across the inner mitochondrial membrane, and furthermore, marker enzyme assays and Western blotting studies exclude any import by contaminating chloroplasts in the preparation. The pea mitochondria specifically recognize information in the chloroplast-targeting presequences, because they also import a fusion comprising the presequence of coproporphyrinogen III oxidase linked to green fluorescent protein. However, the same construct is targeted exclusively into chloroplasts in vivo indicating that the in vitro mitochondrial import reactions are unphysiological, possibly because essential specificity factors are absent in these assays. Finally, we show that disruption of potential amphipathic helices in one presequence does not block import into pea mitochondria, indicating that other features are recognized.
Cleary
Suzanne P
SP
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom.
Tan
Fui-Ching
FC
Nakrieko
Kerry-Ann
KA
Thompson
Simon J
SJ
Mullineaux
Philip M
PM
Creissen
Gary P
GP
von Stedingk
Erik
E
Glaser
Elzbieta
E
Smith
Alison G
AG
Robinson
Colin
C
D029681:Arabidopsis Proteins; D015153:Blotting, Western; D002734:Chlorophyll / Q000737:chemistry; D002736:Chloroplasts / Q000737:chemistry / Q000378:metabolism*; D003304:Coproporphyrinogen Oxidase / Q000737:chemistry; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism*; D018532:Pisum sativum; D018521:Plant Physiological Phenomena; D011498:Protein Precursors / Q000737:chemistry; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism
D029681:Arabidopsis Proteins; C115034:HCF136 protein, Arabidopsis; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D002734:Chlorophyll; D049452:Green Fluorescent Proteins; D003304:Coproporphyrinogen Oxidase