Mitochondrial gene knockout (rho(0)) cells that depend on glycolysis for their energy requirements show an increased ability to reduce cell-impermeable tetrazolium dyes by electron transport across the plasma membrane. In this report, we show for the first time, that oxygen functions as a terminal electron acceptor for trans-plasma membrane electron transport (tPMET) in HL60rho(0) cells, and that this cell surface oxygen consumption is associated with oxygen-dependent cell growth in the absence of mitochondrial electron transport function. Non-mitochondrial oxygen consumption by HL60rho(0) cells was extensively inhibited by extracellular NADH and NADPH, but not by NAD(+), localizing this process at the cell surface. Mitochondrial electron transport inhibitors and the uncoupler, FCCP, did not affect oxygen consumption by HL60rho(0) cells. Inhibitors of glucose uptake and glycolysis, the ubiquinone redox cycle inhibitors, capsaicin and resiniferatoxin, the flavin centre inhibitor, diphenyleneiodonium, and the NQO1 inhibitor, dicoumarol, all inhibited oxygen consumption by HL60rho(0) cells. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and reduction of the cell-impermeable tetrazolium dye, WST-1, suggest that both systems may share a common tPMET pathway. This is supported by the finding that terminal electron acceptors from both pathways compete for electrons from intracellular NADH.

The function of the decoding release factor (RF) in translation termination is to couple cognate recognition of the stop codon in the mRNA with hydrolysis of the completed polypeptide from its covalently linked tRNA. For this to occur, the RF must interact with specific A-site components of the active centers within both the small and large ribosomal subunits. In this work, we have used directed hydroxyl radical footprinting to map the ribosomal binding site of the Escherichia coli class I release factor RF2, during translation termination. In the presence of the cognate UGA stop codon, residues flanking the universally conserved 250 GGQ 252 motif of RF2 were each shown to footprint to the large ribosomal subunit, specifically to conserved elements of the peptidyltransferase and GTPase-associated centers. In contrast, residues that flank the putative "peptide anticodon" of RF2, 205 SPF 207 , were shown to make a footprint in the small ribosomal subunit at positions within well characterized 16 S rRNA motifs in the vicinity of the decoding center. Within the recently solved crystal structure of E. coli RF2, the GGQ and SPF motifs are separated by 23 Å only, a distance that is incompatible with the observed cleavage sites that are up to 100 Å apart. Our data suggest that RF2 may undergo gross conformational changes upon ribosome binding, the implications of which are discussed in terms of the mechanism of RF-mediated termination.Termination of protein synthesis is signaled by the translocation of a stop rather than a sense codon into the A-site of the ribosome (1, 2). The stop codon is recognized by a protein, the polypeptide chain release factor (RF), 1 which triggers the hydrolytic release of the nascent polypeptide chain from the Psite-bound peptidyl-tRNA. Recognition of the stop codon by a protein rather than a tRNA molecule led Moffat and Tate (3) to propose the "tRNA analogue model," which describes the RF as a decoding molecule like tRNA. Class I RFs are the decoding factors (4). In bacteria such as Escherichia coli, there are two decoding RFs, RF1 (decodes UAG and UAA) and RF2 (decodes UGA and UAA). In eukaryotes, a single factor, eRF1, recognizes all three stop codons (5). With the exception of a universally conserved GGQ motif, there is no overall sequence homology between the eubacterial class I RFs and their eukaryotic counterparts (6). The properties of the class I RFs are remarkably similar to those of aminoacyl-tRNAs (reviewed in Ref. 7). RFs compete with suppressor tRNAs for binding to the A-site of the ribosome in response to specific recognition of the A-site codon (8, 9). This so-called anti-suppressor phenotype implies that tRNAs and RFs are cognate species in direct competition for stop codon binding. Both interact with the peptidyl-tRNA located at the ribosomal P-site, and both interact on the ribosome with translational G proteins (EF-Tu for aminoacyl-tRNA and RF3/eRF3 for the decoding RFs).In the tRNA analogue model (3), the decoding RF was proposed to consist of two functionally separate d...

Reduction of the cell-impermeable tetrazolium salt WST-1 has been used to characterise two plasma membrane NADH oxidoreductase activities in human cells. The trans activity, measured with WST-1 and the intermediate electron acceptor mPMS, utilises reducing equivalents from intracellular sources, while the surface activity, measured with WST-1 and extracellular NADH, is independent of intracellular metabolism. Whether these two activities involve distinct proteins or are inherent to a single protein is unclear. In this work, we have attempted to address this question by examining the relationship between the trans and surface WST-1-reducing activities and a third well-characterised family of cell surface oxidases, the ECTO-NOX proteins. Using blue native-polyacrylamide gel electrophoresis, we have identified a complex in the plasma membranes of human 143B osteosarcoma cells responsible for the NADH-dependent reduction of WST-1. The dye-reducing activity of the 300 kDa complex was attributed to a 70 kDa NADH oxidoreductase activity that cross-reacted with antisera against the ECTO-NOX protein CNOX. Differences in enzyme activities and inhibitor profiles between the WST-1-reducing NADH oxidoreductase enzyme in the presence of NADH or mPMS and the ECTO-NOX family are reconciled in terms of the different purification methods and assay systems used to study these proteins.

Plasma membrane electron transport (tPMET) pathways have been identified in all living cells, and a wide variety of tools have been used to study these processes. In our laboratory we have used the cell-impermeable tetrazolium dye WST-1, together with the mitochondrial gene knockout (rho0) cell model, to investigate one of these pathways. We have shown that growth of HL60rho0 cells is dependent on oxygen, and that these cells consume oxygen at the cell surface. Similarities in inhibition profiles between non-mitochondrial oxygen consumption and WST-1 reduction suggest that both systems share a common tPMET pathway. In support of this, oxygen was shown to compete with the intermediate electron acceptor that mediates WST-1 reduction, for reducing electrons. The observation that tPMET activity is higher in rho0 cells compared to their mitochondrially-competent counterparts was shown to be the result of competition between the mitochondrial and plasma membrane electron transport systems for intracellular reducing equivalents. Elevated rates of dye reduction appear to be mediated through increased expression of the key components of tPMET, which include the cell surface NADH oxidase, CNOX. These findings have played a critical role in shaping our current understanding of the mechanisms of this particular pathway of tPMET.

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