A Soluble Secretory Reporter System in Trypanosoma brucei

doi:10.1074/jbc.271.31.18387

A Soluble Secretory Reporter System in Trypanosoma brucei

STUDIES ON ENDOPLASMIC RETICULUM TARGETING*

From the Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, Madison, Wisconsin 53706

^‡ To whom correspondence should be addressed:
Dept. of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 1300 University Ave., Madison, WI 53706
. E-mail: bangs{at}macc.wisc.edu

Abstract

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 (t_1/2 ~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.

Footnotes

↵^§ Supported by National Institutes of Health Cellular and Molecular Parasitology Training Grant AI07414-04.
↵* This work was supported in part by National Institutes of Health Grant AI35739-02 and American Cancer Society Institutional Research Grant 35-35-5 (to J. D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

VSG

variant surface glycoprotein

GPI

glycosylphosphatidylinositol

ER

endoplasmic reticulum

BiP

binding protein

PBS

phosphate-buffered saline

pBSIISK

Bluescript II SK-,PARP, procyclic acidic repetitive protein

CAT

chloramphenicol acetyltransferase

Neo

neomycin phosphotransferase

PCR

polymerase chain reaction

GUS

β-glucaronidase

hsp70

heat shock protein 70 kDa, bp base pair(s).
↵² J. D. Bangs, E. M. Brouch, D. M. Ransom, and J. L. Roggy, unpublished observations.
- Received February 29, 1996.
- Revision received May 14, 1996.