Peroxisomes, luciferase

A novel peroxisome proliferator-activated receptor responsive element-luciferase reporter mouse reveals gender specificity of peroxisome proliferator-activated receptor activity in liver. Mol Endocrinol. 21(2), 388-400. 

Ciana, P., Bisemi, A., Tatangelo, L., Tiveron, C., Sciarroni, A. R, Ottobrini, L., and Maggi, A. (2007) A novel peroxisome prohferator-activated receptor responsive element-luciferase reporter mouse reveals gender specificity of peroxisome prohferator-activated receptor activity in fiver. Mol. Endocrinol. 21, 388 400. 

Recently sequences which can route proteins to peroxisomes have been identified (91). Firefly luciferase, a peroxisomal protein, contains a C-terminal serine-lysine-leucine which was shown to be critical for proper targeting. Indeed, most peroxisomal proteins analyzed thus far show similar sequences at their C-terminus a minority of proteins apparently utilize amino-terminal or internal signal sequences (92,93). 

Approximately 2.4kb of the promoter region of CTE-I and 2.8kb of the promoter region of MTE-I were sequenced in the 5 direction from the ATG start site. The promoter sequences were analysed for the presence of various transcription factor sites, using TESS String Based Search, which identified putative peroxisome proliferator response elements (PPREs) in both promoters. These regions of the promoters have now been cloned into a luciferase expression vector, to be used in transfection e5q)eriments to examine for functional PPREs. 

In mammalian cells transfected with a luciferase-expressing plasmid, we showed by indirect immunofluorescence that luciferase is localized to small vesicular structures (24). Subsequent work involving double-immunolabeling experiments revealed that these vesicles were peroxisomes (37). Interestingly, luciferase is also peroxisomal in the firefly lantern, in yeast and in plant cells... 

FIGURE I Permeabilization of CHO cells with SLO and reconstitution of peroxisomal protein import. Cells were treated with (A) or without (B) 0.2 U/ml SLO, fixed, and incubated with antitubulin antibodies followed by rhodamine-coupled secondary antibodies. Cells per-meabilized with 0.2 U/ml SLO were incubated for 45 min at 37°C with HSA-SKL (C) or luciferase (D), and the localization of the exogenously added substrates was analyzed by immunofluorescence. Bar = 20 /xm. Reprinted from the Journal of Cell Biology, 1993, Vol. 120, pp. 675-685, by copyright permission of the Rockefeller University Press. 

The firefly enzyme might have its own problem by the fact that it is localized in vivo (yeast, mammalian, and plant cells as in the firefly lantern) in small vesicular structures called peroxisomes (55). This is due to the presence of a peroxisomal translocation signal located at the C-terminal domain of the molecule. The peroxisomal localization may present an additional membrane barrier for in situ detection of the enzyme, although it offers a particular advantage for studying protein transport or targeting to peroxisomes. Removal of the peroxisomal translocation signal has been shown to provide an alternative system in which the modified firefly luciferase is expressed as a cytoplasmic enzyme like bacterial luciferases. 

Firefly luciferase from P. pyralis is a 50-kDa single polypeptide (550 amino acids). A C-terminal tripeptide sequence, —Ser—Lys—Leu, serves as the peroxisomal translocation signal, and its removal abolishes import of the enzyme into peroxisomes. Luc is hardly soluble in water, and its solubilization requires some salt. In solvents of relatively low ionic strengths, the protein aggregates in a rapidly reversible manner as the solubility limit is approached (69). 

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