4.5 Article

Defective mitochondrial translation differently affects the live cell dynamics of complex I subunits

期刊

BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS
卷 1807, 期 12, 页码 1624-1633

出版社

ELSEVIER
DOI: 10.1016/j.bbabio.2011.09.013

关键词

Doxycycline; Mitochondria; FRAP; AcGFP1; Fluorescence gel analysis; Chloramphenicol

资金

  1. Radboud University Nijmegen Medical Centre (RUNMC)
  2. ZON (Netherlands Organization for Health Research and Development) [903-46-176]
  3. NWO (Netherlands Organization for Scientific Research) [911-02-008]
  4. CSBR (Centres for Systems Biology Research) initiative from the Nederlandse organisatie voor Wetenschappelijk Onderzoek (NWO, Netherlands Organisation for Scientific Research) [CSBR09/013V]

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Complex I (Cl) of the oxidative phosphorylation system is assembled from 45 subunits encoded by both the mitochondrial and nuclear DNA. Defective mitochondrial translation is a major cause of mitochondrial disorders and proper understanding of its mechanisms and consequences is fundamental to rational treatment design. Here, we used a live cell approach to assess its consequences on CI assembly. The approach consisted of fluorescence recovery after photobleaching (FRAP) imaging of the effect of mitochondrial translation inhibition by chloramphenicol (CAP) on the dynamics of AcGFP1-tagged CI subunits NDUFV1, NDUFS3, NDUFA2 and NDUFB6 and assembly factor NDUFAF4. CAP increased the mobile fraction of the subunits, but not NDUFAF4, and decreased the amount of CI, demonstrating that CI is relatively immobile and does not associate with NDUFAF4. CAP increased the recovery kinetics of NDUFV1-AcGFP1 to the same value as obtained with AcGFP1 alone, indicative of the removal of unbound NDUFV1 from the mitochondrial matrix. Conversely, CAP decreased the mobility of NDUFS3-AcGFP1 and, to a lesser extent, NDUFB6-AcGFP1, suggestive of their enrichment in less mobile subassemblies. Little, if any, change in mobility of NDUFA2-AcGFP1 could be detected, suggesting that the dynamics of this accessory subunit of the matrix arm remains unaltered. Finally, CAP increased the mobility of NDUFAF4-AcGFP1, indicative of interaction with a more mobile membrane-bound subassembly. Our results show that the protein interactions of CI subunits and assembly factors are differently altered when mitochondrial translation is defective. (C) 2011 Elsevier B.V. All rights reserved.

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