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J. Biol. Chem., Vol. 280, Issue 10, 9030-9036, March 11, 2005

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Probing the Role of Compartmentation of Glycolysis in Procyclic Form Trypanosoma brucei

RNA INTERFERENCE STUDIES OF PEX14, HEXOKINASE, AND PHOSPHOFRUCTOKINASE^*

Peter S. Kessler and Marilyn Parsons

From the Seattle Biomedical Research Institute, Seattle, Washington 98109 and the Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98195

Trypanosoma brucei and related organisms contain an organelle evolutionarily related to peroxisomes that sequesters glycolysis, among other pathways. We have shown previously that disruption of protein import into this organelle, the glycosome, can be accomplished through RNA interference (RNAi)-mediated knockdown of the peroxin PEX14. Decreased PEX14 in turn leads to cell death, which, at least in the procyclic stage, can be triggered by the presence of glucose. Here we show that fructose, which is taken up and metabolized by procyclic form T. brucei, and glycerol, which interfaces with the glycosomal glycolytic pathway, are also toxic during PEX14 RNAi. Earlier computer modeling studies predicted that glycolysis would be toxic to T. brucei in the absence of glycosomal compartmentation because of the intrinsic lack of feedback regulation of the parasite hexokinase and phosphofructokinase. To further test this hypothesis, we performed double RNAi, targeting hexokinase and PEX14. Knockdown of hexokinase rescued PEX14 knockdown cells from glucose toxicity, even though glycosomal proteins continue to be mislocalized to the cytosol. Knockdown of phosphofructokinase was benign in the absence of glucose but toxic in the presence of glucose. When PEX14 and phosphofructokinase mRNAs were jointly targeted for RNAi, glycerol remained toxic to the parasites. Taken together, these data indicate that the glycosome provides significant, but not complete, protection of trypanosomes from the dangerous design of glycolysis.

Received for publication, October 25, 2004 , and in revised form, January 3, 2005.

^* This work was supported in part by National Institutes of Health Grant R01 AI22635 and the M. J. Murdock Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Seattle Biomedical Research Inst., 307 Westlake Ave. N. STE 500, Seattle, WA 98109. Tel.: 206-256-7315; Fax: 206-256-7229; E-mail: marilyn.parsons{at}sbri.org.

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