Characterization of an in vitro assay for import of 3-phosphoglycerate kinase into the glycosomes of Trypanosoma brucei.
Glycosomes are microbody organelles found in kinetoplastida, where they serve to compartmentalize the enzymes of the glycolytic pathway. In order to identify the mechanism by which these enzymes are targeted to the glycosome, we have modified the in vitro import assay developed by Dovey et al. (Proc. Natl. Acad. Sci. USA 85:2598-2602, 1988). This assay measures the uptake of in vitro-translated Trypanosoma brucei glycosomal 3-phosphoglycerate kinase (gPGK) by purified glycosomes. Up to 50% of the total 35S-gPGK in the glycosomal fraction was resistant to extraction by 3 M urea or treatment with proteinase K (500 micrograms/ml). The glycosome-associated 35S-gPGK could be chemically cross-linked to the endogenous glycosomal proteins to form a sodium dodecyl sulfate-resistant complex, suggesting that it is close to the intraglycosomal protein matrix. Deoxycholate solubilized the glycosome and thereby rendered the glycosome-associated 35S-gPGK fully susceptible to proteinase K. However, the glycosome-associated 35S-gPGK was not digested by proteinase K in the presence of Triton X-100, which cannot dissolve the glycosomal protein core. The 35S-gPGK synthesized in vitro was able to bind directly to protein cores, where it became resistant to urea extraction and proteinase K digestion. However, the 35S-gPGK-protein core complex exhibited a much higher density than the 35S-gPGK-glycosome complex and was readily separable in sucrose gradients. Thus, in our in vitro import assay, the 35S-gPGK appeared to associate with intact glycosomes, possibly reflecting import of protein into the organelle. Complete denaturation of the 35S-gPGK in 8 M urea prior to the assay enhanced the efficiency of its association with glycosomes. Native gPGK did not compete with the association of in vitro-translated gPGK unless it was denatured. The assay exhibited time and temperature dependence, but it did not require externally added ATP and was not inhibited by the nonhydrolyzable analogs adenosine-5'-(beta,gamma-imido)-triphosphate and gamma-S-ATP. However, the presence of 20 to 30 microM ATP inside the glycosome may fulfill the requirement for protein import.
Sommer
J M
JM
Department of Pharmaceutical Chemistry, University of California, San Francisco 94143.
Thissen
J A
JA
Parsons
M
M
Wang
C C
CC
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008830:Microbodies / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D014346:Trypanosoma brucei brucei / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D014508:Urea
D014508:Urea; D010735:Phosphoglycerate Kinase