In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.

Abstract

The compartmentalization of glycolytic enzymes into specialized organelles, the glycosomes, allows the bloodstream form of Trypanosoma brucei to rely solely on glycolysis for its energy production. The biogenesis of glycosomes in these parasites has been studied intensively as a potential target for chemotherapy. We have adapted the recently developed methods for stable transformation of T. brucei to the in vivo analysis of glycosomal protein import. Firefly luciferase, a peroxisomal protein in the lantern of the insect, was expressed in stable transformants of the procyclic form of T. brucei, where it was found to accumulate inside the glycosomes. Mutational analysis of the peroxisomal targeting signal serine-lysine-leucine (SKL) located at the C-terminus of luciferase showed that replacement of the serine residue (Serine548) with a small neutral amino acid (A, C, G, H, N, P, T) still resulted in an import efficiency of 50-100% of the wild-type luciferase. Lysine549 could be substituted with an amino acid capable of hydrogen bonding (H, M, N, Q, R, S), whereas the C-terminal leucine550 could be replaced with a subset of hydrophobic amino acids (I, M, Y). Thus, a peroxisome-like C-terminal SKL-dependent targeting mechanism may function in T. brucei to import luciferase into the glycosomes. However, a few significant differences exist between the glycosomal targeting signals identified here and the tripeptide sequences that direct proteins to mammalian or yeast peroxisomes.

Interferon-γ Activation of a Mitogen-activated Protein Kinase, KFR1, in the Bloodstream Form of Trypanosoma brucei

“…The orientation and nucleotide sequence of KFR1 in pTKH-Hyg were confirmed by sequencing. Expression of both KFR1 and hyg r is under the control of the PARP promoter, and there is a splicing signal site upstream of each open reading frame (22). Purified plasmid DNA of pTKH-Hyg was linearized at the MluI site in the trypanosome tubulin intergenic region in the plasmid and transformed into procyclic T. brucei cells by electroporation (22).…”

“…Expression of both KFR1 and hyg r is under the control of the PARP promoter, and there is a splicing signal site upstream of each open reading frame (22). Purified plasmid DNA of pTKH-Hyg was linearized at the MluI site in the trypanosome tubulin intergenic region in the plasmid and transformed into procyclic T. brucei cells by electroporation (22). After stable transformants were selected with 50 g/ml hygromycin B (Calbiochem), cell lines were cloned by limiting dilution (22).…”

“…Purified plasmid DNA of pTKH-Hyg was linearized at the MluI site in the trypanosome tubulin intergenic region in the plasmid and transformed into procyclic T. brucei cells by electroporation (22). After stable transformants were selected with 50 g/ml hygromycin B (Calbiochem), cell lines were cloned by limiting dilution (22). Expression of KFR1 was examined at mRNA and protein levels by Northern hybridization and Western blotting, respectively, as described (16).…”

“…Likewise, a fragment containing the PARP promoter and neo r was released from pTSA-NEO2 (22) with SacI and T4 DNA polymerase and followed by a partial digestion of BssHII. Due to the presence of BssHII site in the open reading frame of neo r , only the fragment containing the complete neo r open reading frame was recovered from an agarose gel.…”

“…Expression of KFR1 and its downstream hyg r gene was controlled by the same constitutive PARP promoter and the polycistronic transcript processed by trans-splicing (27). Stable transfectants were selected under hygromycin B and were subsequently cloned by limiting dilution (22). Northern hybridizations indicated that the transfected KFR1 gene was successfully transcribed (Fig.…”