Identification of Mitochondrial Complex I Assembly Intermediates by Tracing Tagged Ndufs3 Demonstrates the Entry Point of Mitochondrial Subunits.
From: Nijmegen Centre for Mitochondrial Disorders, Department of Paediatrics, Radboud University Nijmegen Medical Centre, 6500 HB Nijmegen, The Netherlands.
The Journal of biological chemistry
- Publish Date: Mar 2007
- ISSN: 0021-9258
- Volume: 282
- Issue: 10
- Pages: 7582-90
- Medium: Print
- Language: English
- Citation (JAMA): Vogel Rutger O, Dieteren Cindy E J, van den Heuvel Lambert P W J, et al. Identification of Mitochondrial Complex I Assembly Intermediates by Tracing Tagged Ndufs3 Demonstrates the Entry Point of Mitochondrial Subunits.. J. Biol. Chem. Mar 2007;282:7582-90
Abstract
Biogenesis of human mitochondrial complex I (CI) requires the coordinated assembly of 45 subunits derived from both the mitochondrial and nuclear genome. The presence of CI subcomplexes in CI-deficient cells suggests that assembly occurs in distinct steps. However, discriminating between products of assembly or instability is problematic. Using an inducible NDUFS3-green fluorescent protein (GFP) expression system in HEK293 cells, we here provide direct evidence for the stepwise assembly of CI. Upon induction, six distinct NDUFS3-GFP-containing subcomplexes gradually appeared on a blue native Western blot also observed in wild type HEK293 mitochondria. Their stability was demonstrated by differential solubilization and heat incubation, which additionally allowed their distinction from specific products of CI instability and breakdown. Inhibition of mitochondrial translation under conditions of steady state labeling resulted in an accumulation of two of the NDUFS3-GFP-containing subcomplexes (100 and 150 kDa) and concomitant disappearance of the fully assembled complex. Lifting inhibition reversed this effect, demonstrating that these two subcomplexes are true assembly intermediates. Composition analysis showed that this event was accompanied by the incorporation of at least one mitochondrial DNA-encoded subunit, thereby revealing the first entry point of these subunits.
Mesh Headings (Keywords): Blotting, Western, Cells, Cultured, DNA, Mitochondrial, Electron Transport Complex I, Green Fluorescent Proteins, Humans, Mitochondria, NADH Dehydrogenase, Protein Subunits
Check for Full Text / PubMed Unique Identifier (PMID): 17209039
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