In vivo import of firefly luciferase into the glycosomes of Trypanosoma brucei and mutational analysis of the C-terminal targeting signal.
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.
Sommer
J M
JM
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Cheng
Q L
QL
Keller
G A
GA
Wang
C C
CC
D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D015870:Gene Expression; D005822:Genetic Vectors; D006019:Glycolysis; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D015388:Organelles / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014346:Trypanosoma brucei brucei / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008156:Luciferases