A soluble secretory reporter system in Trypanosoma brucei. Studies on endoplasmic reticulum targeting.
A homolog of the endoplasmic reticulum (ER) hsp70 protein, binding protein (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-precipitation experiments, BiP transiently associates with newly synthesized 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. Deletion of the BiP C-terminal tetrapeptide (MDDL) and the glycosylphosphatidylinositol-anchor addition sequence of VSG converts these proteins to secreted forms. Attachment of MDDL to VSG results in intracellular retention 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 approximately 1 h) of the N-terminal ATPase domain (BiPN), consistent with the conserved domain structure of hsp70 proteins. This is the first demonstration of soluble protein secretion in African trypanosomes. Using the BiPN reporter, the sequence specificity of C-terminal tetrapeptide 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 throughout the wide range of eukaryotic evolution.
Bangs
J D
JD
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, Madison, Wisconsin 53706, USA.
Brouch
E M
EM
Ransom
D M
DM
Roggy
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017930:Genes, Reporter; D005822:Genetic Vectors; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D017931:DNA Primers; D016054:DNA, Protozoan; D018840:HSP70 Heat-Shock Proteins; D021382:Protein Sorting Signals; D015800:Protozoan Proteins