Rhomboid proteases: familiar features in unfamiliar phases.

In this issue of Molecular Cell, Strisovsky et al. (2009) identify a sequence motif underlying cleavage site specificity for the rhomboid proteases. This sheds light on potential mechanisms by which intramembrane-cleaving proteases cleave their substrates.

Transmembrane topology of subunit N of complex I (NADH:ubiquinone oxidoreductase) from Escherichia coli.

The transmembrane topology of subunit N from E. coli Complex I has been investigated. Chemical labeling of mono-substituted cysteine mutants was carried out in inverted membrane vesicles, and in whole cells, using 3-N-maleimidyl-propionyl biocytin (MPB). The results support a model of 14 transmembrane spans with both termini in the periplasm, and are consistent with the models of subunits L, M and N from the crystal structure of the membrane arm of the E. coli Complex I (Efremov et al. (2010) Nature 465, 441-445). In particular, the results do not support an unusual cytoplasmic localization of two likely transmembrane regions, as proposed in previous studies (Mathiesen and Hägerhäll (2002) Biochim Biophys Acta 1556, 121-132; Torres-Bacete, et al. (2009) J Biol Chem 284, 33062-33069).

Construction of a deletion strain and expression vector for the Escherichia coli NADH:ubiquinone oxidoreductase (Complex I).

Complex I of Escherichia coli is encoded by 13 consecutive genes, called the nuo operon. A chromosomal deletion of all nuo genes has been achieved by homologous recombination. A vector that encodes all of the nuo genes has been constructed, and it expresses a functional enzyme.

Direct transfer of NADH from malate dehydrogenase to complex I in Escherichia coli.

During aerobic growth of Escherichia coli, nicotinamide adenine dinucleotide (NADH) can initiate electron transport at either of two sites: Complex I (NDH-1 or NADH:ubiquinone oxidoreductase) or a single-subunit NADH dehydrogenase (NDH-2). We report evidence for the specific coupling of malate dehydrogenase to Complex I. Membrane vesicles prepared from wild type cultures retain malate dehydrogenase and are capable of proton translocation driven by the addition of malate + NAD. This activity was inhibited by capsaicin, an inhibitor specific to Complex I, and it proceeded with deamino-NAD, a substrate utilized by Complex I, but not by NDH-2. The concentration of free NADH produced by membrane vesicles supplemented with malate + NAD was estimated to be 1 microM, while the rate of proton translocation due to Complex I was consistent with a somewhat higher concentration, suggesting a direct transfer mechanism. This interpretation was supported by competition assays in which inactive mutant forms of malate dehydrogenase were able to inhibit Complex I activity. These two lines of evidence indicate that the direct transfer of NADH from malate dehydrogenase to Complex I can occur in the E. coli system.

Activation of sterol regulatory element-binding protein by the caspase Drice in Drosophila larvae.

During larval development in Drosophila melanogaster, transcriptional activation of target genes by sterol regulatory element-binding protein (dSREBP) is essential for survival. In all cases studied to date, activation of SREBPs requires sequential proteolysis of the membrane-bound precursor by site-1 protease (S1P) and site-2 protease (S2P). Cleavage by S2P, within the first membrane-spanning helix of SREBP, releases the transcription factor. In contrast to flies lacking dSREBP, flies lacking dS2P are viable. The Drosophila effector caspase Drice cleaves dSREBP, and cleavage requires an Asp residue at position 386, in the cytoplasmic juxtamembrane stalk. The initiator caspase Dronc does not cleave dSREBP, but animals lacking dS2P require both drice and dronc to complete development. They do not require Dcp1, although this effector caspase also can cleave dSREBP in vitro. Cleavage of dSREBP by Drice releases the amino-terminal transcription factor domain of dSREBP to travel to the nucleus where it mediates the increased transcription of target genes needed for lipid synthesis and uptake. Drice-dependent activation of dSREBP explains why flies lacking dS2P are viable, and flies lacking dSREBP itself are not.

Mutagenesis of subunit N of the Escherichia coli complex I. Identification of the initiation codon and the sensitivity of mutants to decylubiquinone.

The last gene in the nuo operon of Escherichia coli, nuoN, encodes a membrane-bound subunit of Complex I (NADH:ubiquinone oxidoreductase). In this report, the gene for subunit N was disrupted by a 163 bp deletion in the chromosome, resulting in the loss of Complex I function, as measured by deamino-NADH oxidase activity. This activity could be recovered after transformation of the mutant strain by a plasmid that contains the previously identified nuoN gene and the upstream intergenic region between nuoM and nuoN. Mutagenesis of the first ATG downstream of nuoM led to a loss of function, indicating that this is the likely initiation codon for nuoN, and predicting a protein of 485 amino acids and 52 044 Da. Thirty site-specific mutations in nuoN at 19 different positions were constructed in a vector that expresses the full-length subunit N with both an octahistidine tag and an HA epitope tag at the carboxyl terminus. Highly conserved charged and aromatic residues were selected for mutagenesis, as well as a substitution that occurs as a secondary mutation in Leber's hereditary optic neuropathy (LHON). Membranes from the mutant strains were tested for production of subunit N by immunoblots and for NADH-linked activities. Mutants with substitutions at six different positions (K158, K217, H224, K247, Y300, and K395) had rates of deamino-NADH oxidase activity that were no more than 50% of that of the wild type and had reduced rates of proton translocation. These mutants also showed enhanced inhibition by decylubiquinone, indicating that subunit N interacts with quinones. The mutation associated with LHON, G391S, had little effect on these functions.