Development and Characterization of a Conditional Mitochondrial Complex I Assembly System

Abstract

We developed a conditional complex I assembly system in a Chinese hamster fibroblast mutant line, CCL16-B2, that does not express the NDUFA1 gene (encoding the MWFE protein). In this mutant, a hemagglutinin (HA) epitope-tagged MWFE protein was expressed from a doxycycline-inducible promoter. The expression of the protein was absolutely dependent on the presence of doxycycline, and the gene could be turned off completely by removal of doxycycline. These experiments demonstrated a key role of MWFE in the pathway of complex I assembly. Upon induction the MWFE⅐HA protein reached steady-state levels within 24 h, but the appearance of fully active complex I was delayed by another ϳ24 h. The MWFE appeared in a precomplex that probably includes one or more subunits encoded by mtDNA. The fate of MWFE and the stability of complex I were themselves very tightly linked to the activity of mitochondrial protein synthesis and to the assembly of subunits encoded by mtDNA (ND1-6 and ND4L). This novel conditional system can shed light not only on the mechanism of complex I assembly but emphasizes the role of subunits previously thought of as "accessory." It promises to have broader applications in the study of cellular energy metabolism and production of reactive oxygen species and related processes.The mammalian mitochondrial complex I (NADH-ubiquinone oxidoreductase) is an assembly of ϳ46 distinct proteins encoded by 39 nuclear and 7 mitochondrial genes (1). Fourteen of these subunits, including all the mtDNA-encoded ones, have orthologues in prokaryotes (2). This simpler prokaryotic complex of 14 subunits can perform all the currently known functions of mammalian complex I: NADH oxidation, electron transport from the flavin mononucleotide via several Fe-S centers to a quinone, and proton translocation across the membrane. The roles of the other 32 "accessory" or "supernumerary proteins" in the mammalian complex I are largely unknown. Many of these subunits are also found in fungal and plant complexes. Only a few of these accessory proteins, e.g. the acyl-carrier protein SDAP and the AQDQ and MWFE subunits from Neurospora crassa and mammals have been studied in more detail (3-5). The orthologue of the mammalian MWFE in N. crassa was identified recently (6). Like its mammalian counterpart it was found to be essential for complex I biogenesis.From bacteria to humans, the overall "boot-shaped" structure of complex I is conserved with one arm protruding into the matrix and the other localized in the plane of the membrane (7-11). The matrix arm contains all the prosthetic groups, whereas the function of the large membrane arm is less clear. Recent findings suggest that it is involved in the binding of quinone and inhibitors and proton/ion translocation across the inner membrane (12). Studies with a N. crassa mutant indicate that complex I assembly takes place in discrete steps with independent assembly of membrane and peripheral arms (13). The attachment of the peripheral arm to the membrane depends upon the assembly of the membrane...

Insights into the Composition and Assembly of the Membrane Arm of Plant Complex I through Analysis of Subcomplexes in Arabidopsis Mutant Lines

“…The role of MWFE would thus be consistent with its use to allow the assembly of a subset of mitochondria-encoded subunits. A similar role for MWFE has been proposed in mammals in a conditional assembly system (43). Three subunits (NDU9, PSST, and MWFE) were only detected in the 650-kDa subcomplex and were absent from the smaller subcomplexes.…”

Mitochondrial Dysfunction Impairs Tumor Suppressor p53 Expression/Function

“…As these Res Ϫ cells completely rely on glycolysis for their bioenergetic needs, they must be grown in the presence of plenty of glucose with frequent change of the culture medium (39 -42). The generation of B2-MWFE i cells using the pTre-On vector has been described recently (35,38). The G18-ESSS i cells were also developed by the same strategy.…”

“…All other reagents were from Sigma unless otherwise stated Cells and Culture Conditions-The respiration-deficient (Res Ϫ ) cells used in this study have been described elsewhere (see Table 1 for details) (35)(36)(37)(38). As these Res Ϫ cells completely rely on glycolysis for their bioenergetic needs, they must be grown in the presence of plenty of glucose with frequent change of the culture medium (39 -42).…”

“…In light of this study, caution must be taken to avoid the chronic presence of antibiotics such as tetracycline (and its derivatives) that can impair mitochondrial protein synthesis in cell culture medium. In inducible cells (B2-MWFE i and G18-ESSS i ), complex I assembly was induced using 1 g/ml doxycycline (Dox) as described previously (35,38). Cells were harvested after washing once with Ca 2ϩ -, Mg 2ϩ -free phosphate-buffered saline (PBS), pH 7.4, using trypsin/EDTA (Invitrogen).…”

“…Reversible-To eliminate the possibility that the p53 gene in Res Ϫ cells may be mutated, we used a conditional complex I assembly system (B2-MWFE i ) recently described by us to determine whether restoring complex I assembly would result in gain-of-p53 function and ␥IR sensitivity (35,38). In B2-MW-FE i cells, complex I assembly was induced with 1 g/ml Dox in the medium for 48 h. Then cells were allowed to recover without Dox for 24 h and treated with 40 Gy ␥IR.…”