A quantitative assay for mitochondrial fusion using Renilla luciferase complementation

Mitochondria continuously undergo fusion and fission, the relative rates of which define their morphology. Large mitochondria produce energy more efficiently, whereas small mitochondria translocate better to subcellular sites where local production of ATP is acutely required. Mitochondrial fusion is currently assayed by fusing together cells expressing GFP or RFP in their mitochondria and then scoring the frequency of cells with yellow mitochondria (representing fused green and red mitochondria). However, this assay is labor-intensive and only semi-quantitative. We describe here a reporter system consisting of split fragments of Renilla luciferase and YFP fused to mitochondrial matrix-targeting sequences and to leucine zippers to trigger dimerization. The assay enables fusion to be quantitated both visually for individual cells and on a population level using chemiluminescence, laying the foundation for high throughput small molecule and RNAi screens for modulators of mitochondrial fusion. We use the assay to examine cytoskeletal roles in fusion progression.     

Purification and properties of Renilla reniformis luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis (Renilla luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5.) catalyzes the bioluminescent oxidation of Renilla luciferin producing light (lambdaB 480 nm, QB 5.5%), oxyluciferin, and CO2 (Hori, K., Wampler, J.E., Matthews, J.C., and Cormier, M.J. (1973), Biochemistry 12, 4463). Using a combination of ion-exchange, molecular-sieve, sulfhydryl-exchange, and affinity chromatography, luciferase has been purified, approximately 12 000-fold with 24% recovery, to homogeneity as judged by analysis with disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and ultracentrifugation. Renilla luciferase is active as a nearly spherical single polypeptide chain monomer of 3.5 X 10(4) daltons having a specific activity of 1.8 X 10(15) hp s-1 mg-1 and a turnover number of 111 mumol min-1 mumol-1 of enzyme. This enzyme has a high content of aromatic and hydrophobic amino acids such that it has an epsilon280nm 0.1% of 2.1 and an average hydrophobicity of 1200 cal residue-1. The high average hydrophobicity of luciferase, which places it among the more hydrophobic proteins reported, is believed to account, at least in part, for its tendency to self-associate forming inactive dimers and higher molecular weight species.     

Substrate and substrate analogue binding properties of Renilla luciferase.

Luciferase from the anthozoan coelenterate Renilla reniformis catalyzes the oxidative decarboxylation of luciferin consuming 1 mol of O2 per mol of luciferin oxidized and producing 1 mol of CO2, 1 mol of oxyluciferin, and light (lambdaB, 480 nm) with a 5.5% quantum yield. In this work we have examined the binding characteristics of luciferin, luciferin analogues, and competitive inhibitors of the luciferin-luciferase reaction. The results show that luciferin binding and orientation in the single luciferin binding site of luciferase are highly specific for and dependent upon the three group substituents of the luciferin molecule while the imidazolone-pyrazine nucleus of luciferin is not directly involved in binding. Anaerobic luciferin binding promotes a rapid concentration-dependent aggregation of luciferase which results in irreversible inactivation of the enzyme. This aggregation phenomenon is not observed upon binding of oxyluciferin, luciferyl sulfate, or luciferin analogues in which the substituent at the 2 position of the imidazolone-pyrazine ring has been substantially altered.     

In vivo testing of Renilla luciferase substrate analogs in an orthotopic murine model of human glioblastoma

In vivo bioluminescent imaging using cells expressing Renilla luciferase is becoming increasingly common. Hindrances to the more widespread use of Renilla luciferase are the high autoluminescence of its natural substrate, coelenterazine, in plasma, the relatively high absorbance by tissue of the light emitted by the enzyme-substrate reaction; rapid clearance of the substrate; and significant cost. These factors, save for the cost, which has its own limiting effect on use, can combine to reduce the sensitivity of in vivo assays utilizing this reporter system, and methods of increasing light output or decreasing autoluminescence could be of great benefit. A number of analogs of coelenterazine are being investigated may accomplish one or both of these goals. In this study that we report on the testing of two new substrate analogs, EnduRen and ViViren, manufactured by Promega Corporation, in an orthotopic murine model of human glioblastoma expressing Renilla luciferase. We have tested these analogs in this cell line both in vitro and in vivo, and find that the substrate viviren results in significantly greater light output than the natural substrate or the other analog EnduRen. This new substrate could be valuable for studies where greater sensitivity is important.     

Crystal structures of the luciferase and green fluorescent protein from Renilla reniformis

Due to its ability to emit light, the luciferase from Renilla reniformis (RLuc) is widely employed in molecular biology as a reporter gene in cell culture experiments and small animal imaging. To accomplish this bioluminescence, the 37-kDa enzyme catalyzes the degradation of its substrate coelenterazine in the presence of molecular oxygen, resulting in the product coelenteramide, carbon dioxide, and the desired photon of light. We successfully crystallized a stabilized variant of this important protein (RLuc8) and herein present the first structures for any coelenterazine-using luciferase. These structures are based on high-resolution data measured to 1.4 Å and demonstrate a classic α/β-hydrolase fold. We also present data of a coelenteramide-bound luciferase and reason that this structure represents a secondary conformational form following shift of the product out of the primary active site. During the course of this work, the structure of the luciferase's accessory green fluorescent protein (RrGFP) was also determined and shown to be highly similar to that of Aequorea victoria GFP.     

Structure-function studies on the active site of the coelenterazine-dependent luciferase from Renilla

Renilla luciferase (RLUC) is a versatile tool for gene expression assays and in vivo biosensor applications, but its catalytic mechanism remains to be elucidated. RLUC is evolutionarily related to the alpha/beta hydrolase family. Its closest known homologs are bacterial dehalogenases, raising the question of how a protein with a hydrolase fold can function as a decarboxylating oxygenase. Molecular docking simulations with the coelenterazine substrate against an RLUC homology model as well as a recently determined RLUC crystal structure were used to build hypotheses to identify functionally important residues, which were subsequently tested by site-directed mutagenesis, heterologous expression, and bioluminescence emission spectroscopy. The data highlighted two triads of residues that are critical for catalysis. The putative catalytic triad residues D120, E144, and H285 bear only limited resemblance to those found in the active site of aequorin, a coelenterazine-utilizing photoprotein, suggesting that the reaction scheme employed by RLUC differs substantially from the one established for aequorin. The role of H285 in catalysis was further supported by inhibition using diethylpyrocarbonate. Multiple substitutions of N53, W121, and P220--three other residues implicated in product binding in the homologous dehalogenase Sphingomonas LinB--also supported their involvement in catalysis. Together with luminescence spectra, our data lead us to propose that the conserved catalytic triad of RLUC is directly involved in the decarboxylation reaction of coelenterazine to produce bioluminescence, while the other active-site residues are used for binding of the substrate.     

Pitfall of an internal control plasmid: response of Renilla luciferase (pRL-TK) plasmid to dihydrotestosterone and dexamethasone

The thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) is commonly used as a control for transfection efficiency in the Dual-Luciferase Reporter Assay System. While investigating hormone response elements in the promoters of the androgen-dependent, epididymis-specific EP2 gene, we found that hormone treatment affected the luciferase activity of pRL-TK-transfected cells. In African Green Monkey Kidney (CV-1) cells, cotransfected transiently with a hormone-responsive promoter-firefly luciferase reporter plasmid and with pRL-TK, Renilla luciferase activity increased in response to dihydrotestosterone (DHT) and decreased in response to dexamethasone (DEX). When a thromboxane synthase promoter Renilla luciferase plasmid (pRL-TS) was used in place of pRL-TK, Renilla luciferase activity remained constant in CV-1 cells treated with DHT but decreased in CV-1 cells treated with DEX. In transfection studies, internal control plasmid expression in response to treatment must be carefully monitored to ensure proper interpretation of normalized results.     

Expression of recombinant Renilla luciferase in transgenic plants results in high levels of light emission

A 970 bp DNA fragment which encodes the luciferase enzyme of the marine soft coral Renilla reniformis was fused to the cauliflower mosaic virus (CaMV) promoter. The construct pPCV702-ruc was transferred into alfalfa protoplasts by Ca-PEG-mediated transformation and into tobacco, tomato and potato plants by Agrobacterium -mediated transformation. The light emission from homogenates of alfalfa protoplasts transformed with pPCV702-ruc was 16-fold higher than that of protoplasts transformed with the same vector carrying the bacterial luxF gene. Application of a 3 µM aequous solution of 2-benzyl luciferin (luciferin) on to calli, leaves, roots and slices of tomato fruits and potato tubers of transformed plants resulted in strong light emission within seconds which could be easily visualized by a photon counting camera. Light emissions obtained from tissue homogenates of tobacco plants containing a single copy of the pPCV702-ruc construct were around 20-fold higher than those from plants carrying multiple copies of the firefly luciferase gene and around 360-fold higher than those from plants transformed with the bacterial luciferase gene. Owing to its high efficiency the Renilla luciferase may become a useful and novel tool for gene expression studies in plants and other systems.     

Improved sensitivity of glutamine synthetase assay and of acyl hydroxamates

Sensitivity of glutamine synthetase assay by the hydroxamate method can be doubled by replacing ferric chloride with ferric nitrate or ferric perchlorate, each with the appropriate acid.     

Coelenterazine-binding protein of Renilla muelleri: cDNA cloning, overexpression, and characterization as a substrate of luciferase.

The Renilla bioluminescent system in vivo is comprised of three proteins--the luciferase, green-fluorescent protein, and coelenterazine-binding protein (CBP), previously called luciferin-binding protein (LBP). This work reports the cloning of the full-size cDNA encoding CBP from soft coral Renilla muelleri, its overexpression and properties of the recombinant protein. The apo-CBP was quantitatively converted to CBP by simple incubation with coelenterazine. The physicochemical properties of this recombinant CBP are determined to be practically the same as those reported for the CBP (LBP) of R. reniformis. CBP is a member of the four-EF-hand Ca(2+)-binding superfamily of proteins with only three of the EF-hand loops having the Ca(2+)-binding consensus sequences. There is weak sequence homology with the Ca(2+)-regulated photoproteins but only as a result of the necessary Ca(2+)-binding loop structure. In combination with Renilla luciferase, addition of only one Ca(2+) is sufficient to release the coelenterazine as a substrate for the luciferase for bioluminescence. This combination of the two proteins generates bioluminescence with higher reaction efficiency than using free coelenterazine alone as the substrate for luciferase. This increased quantum yield, a difference of bioluminescence spectra, and markedly different kinetics, implicate that a CBP-luciferase complex might be involved.     

Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals

In vivo imaging of bioluminescent reporters relies on expression of light-emitting enzymes, luciferases, and delivery of chemical substrates to expressing cells. Coelenterazine (CLZN) is the substrate for a group of bioluminescent enzymes obtained from marine organisms. At present, there are more than 10 commercially available CLZN analogs. To determine which analog is most suitable for activity measurements in live cells and living animals, we characterized 10 CLZN analogs using Renilla luciferase (Rluc) as the reporter enzyme. For each analog, we monitored enzyme activity, auto-oxidation, and efficiency of cellular uptake. All CLZN analogs tested showed higher auto-oxidation signals in serum than was observed in phosphate buffer or medium, mainly as a result of auto-oxidation by binding to albumin. CLZN-f, -h, and -e analogs showed 4- to 8-fold greater Rluc activity, relative to CLZN-native, in cells expressing the enzyme from a stable integrant. In studies using living mice expressing Rluc in hepatocytes, administration of CLZN-e and -native produced the highest signal. Furthermore, distinct temporal differences in signal for each analog were revealed following intravenous or intraperitoneal delivery. We conclude that the CLZN analogs that are presently available vary with respect to hRluc utilization in culture and in vivo, and that the effective use of CLZN-utilizing enzymes in living animals depends on the selection of an appropriate substrate.