A SOLUBLE SECRETORY REPORTER SYSTEM IN TRYPANOSOMA-BRUCEI - STUDIES ON ENDOPLASMIC-RETICULUM TARGETING

A homolog of the endoplasmic reticulum (ER) hsp70 protein, binding pro tein (BiP), from the parasitic protozoan Trypanosoma brucei (Bangs, J. D., Uyetake, L., Brickman, M. J., Balber, A. E., and Boothroyd, J. C. (1993) J. Cell Sci, 105, 1101-1113) is further characterized, In co-p recipitation experiments, BiP transiently associates with newly synthe sized secretory proteins, including variant surface glycoprotein (VSG) , confirming its role as a molecular chaperone, To study the molecular signals targeting BiP to the ER, we have developed soluble secretory reporters for expression in transformed procyclic trypanosomes. Deleti on of the BiP C-terminal tetrapeptide (MDDL) and the glycosylphosphati dylinositol-anchor addition sequence of VSG converts these proteins to secreted forms, Attachment of MDDL to VSG results in intracellular re tention confirming that MDDL is a trypanosomal ER localization signal, Secretion of both reporters is inefficient, but further truncation of the BiP C-terminal peptide binding domain allows quantitative export (t1/2 similar to 1 h) of the N-terminal ATPase domain (BiPN), consiste nt with the conserved domain structure of hsp70 proteins, This is the first demonstration of soluble protein secretion in African trypanosom es. Using the BiPN reporter, the sequence specificity of C-terminal te trapeptide retention signals in trypanosomes is analyzed and found to be similar to higher eukaryotes. These results indicate that the basic signals mediating protein targeting to the ER lumen are conserved thr oughout the wide range of eukaryotic evolution.