title: Phosphoenolpyruvate carboxykinase of Trypanosoma brucei is targeted to the glycosomes by a C-terminal sequence.
abstract: Import of proteins into the glycosomes of T. 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 T. brucei PEPCK. However, we found that this peroxisomal targeting signal does not function for glycosomal protein import in T. 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 T. 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 T. brucei PEPCK may contain a second, internal glycosomal targeting signal upstream of the C-terminal SRL sequence.
authors.affiliation: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143-0446.
authors.forename: J M
authors.initials: JM
authors.lastname: Sommer
authors.forename: T T
authors.initials: TT
authors.lastname: Nguyen
authors.forename: C C
authors.initials: CC
authors.lastname: Wang
mesh_terms: D000101:Acetyl-CoA C-Acetyltransferase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism*; D019764:Phosphoenolpyruvate Carboxykinase (ATP); D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000201:enzymology*
chemical_list: D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D008156:Luciferases; D000101:Acetyl-CoA C-Acetyltransferase; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D019764:Phosphoenolpyruvate Carboxykinase (ATP)