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Glycosomal phosphoglycerate kinase (gPGK) of Trvpanosoma brucei differs from the cytoplasmic isozyme (cPGK) in its higher isoelectric point characterized by clusters of positive charges along the polypeptide chain, and a 20 amino acid C-terminal extension ending in serine-serine-leucine (SSL). While a C-terminal SSL tripeptide is apparently not capable of directing luciferase to the peroxisomes in mammalian cells [J, Cell Biol. 108 (1989), 1657-1664], we show here that it is sufficient for the import ofluciferase as well as an unrelated protein, fl-glucuronidase, into the glycosomes of 72 brucei, as determined by immunoelectron microscopy. The analysis of luciferase gPGK fusion proteins indicates that the only targeting signal for import of gPGK into the glycosome resides in this C-terminal SSL sequence.

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Import of proteins into the glycosomes of 7Y brucei resembles the peroxisomal protein import in that C-terminal SKL-like tripeptide sequences can function as targeting signals. Many of the glycosomal proteins do not, however, possess such C-terminal tripeptide signals. Among these, phosphoenolpyruvate carboxykinase (PEPCK (ATP)) was thought to be targeted to the glycosomes by an N-terminal or an internal targeting signal. A limited similarity to the N-terminal targeting signal of rat peroxisomal thiolase exists at the N-terminus of 7: brucei PEPCK. However, we found that this peroxisomal targeting signal does not function for glycosomal protein import in Z brucei. Further studies of the PEPCK gene revealed that the C-terminus of the predicted protein does not correspond to the previously deduced protein sequence of 472 amino acids due to a -1 frame shift error in the original DNA sequence. Readjusting the reading frame of the sequence results in a predicted protein of 525 amino acids in length ending in a tripeptide serine-arginine-leucine (SRL), which is a potential targeting signal for import into the glycosomes. A fusion protein of firefly luciferase, without its own C-terminal SKL targeting signal, and 7: brucei PEPCK is efficiently imported into the glycosomes when expressed in procyclic trypanosomes. Deletion of the C-terminal SRL tripeptide or the last 29 amino acids of PEPCK reduced the import only by about 50% while a deletion of the last 47 amino acids completely abolished the import. These results suggest that 7: brucei PEPCK may contain a second, internal glycosomal targeting signal upstream of the C-terminal SRL sequence.

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