title: A co-translational model to explain the in vivo import of proteins into HeLa cell mitochondria.
abstract: The dual signal approach, i.e. a mitochondrial signal at the N-terminus and an ER (endoplasmic reticulum) or a peroxisomal signal at the C-terminus of EGFP (enhanced green fluorescent protein), was employed in transfected HeLa cells to test for a co-translational import model. The signal peptide from OTC (ornithine transcarbamylase) or arginase II was fused to the N-terminus of EGFP, and an ER or peroxisomal signal was fused to its C-terminus. The rationale was that if the free preprotein remained in the cytosol, it could be distributed between the two organelles by using a post-translational pathway. The resulting fusion proteins were imported exclusively into mitochondria, suggesting that co-translational import occurred. Native preALDH (precursor of rat liver mitochondrial aldehyde dehydrogenase), preOTC and rhodanese, each with the addition of a C-terminal ER or peroxisomal signal, were also translocated only to the mitochondria, again showing that a co-translational import pathway exists for these native proteins. Import of preALDH(sp)-DHFR, a fusion protein consisting of the leader sequence (signal peptide) of preALDH fused to DHFR (dihydrofolate reductase), was studied in the presence of methotrexate, a substrate analogue for DHFR. It was found that 70% of the preALDH(sp)-DHFR was imported into mitochondria in the presence of methotrexate, implying that 70% of the protein utilized the co-translational import pathway and 30% used the post-translational import pathway. Thus it appears that co-translational import is a major pathway for mitochondrial protein import. A model is proposed to explain how competition between binding factors could influence whether or not a cytosolic carrier protein, such as DHFR, uses the co- or post-translational import pathway.
authors.affiliation: Department of Biochemistry, Purdue University, 175 S. University Street, West Lafayette, IN 47907-2063, USA.
authors.forename: Abhijit
authors.initials: A
authors.lastname: Mukhopadhyay
authors.forename: Li
authors.initials: L
authors.lastname: Ni
authors.forename: Henry
authors.initials: H
authors.lastname: Weiner
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000378:metabolism; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells / Q000737:chemistry* / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008727:Methotrexate / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000201:enzymology / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000502:physiology; D008957:Models, Genetic; D008970:Molecular Weight; D020675:Peroxisomes / Q000737:chemistry; D014176:Protein Biosynthesis / Q000235:genetics*; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D021381:Protein Transport / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D024101:Mitochondrial Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000444:Aldehyde Dehydrogenase; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate