Glycolysis modulates trypanosome glycoprotein expression as revealed by an RNAi library.
RNA interference (RNAi) is a powerful tool for identifying gene function in Trypanosoma brucei. We generated an RNAi library, the first of its kind in any organism, by ligation of genomic fragments into the vector pZJMbeta. After transfection at approximately 5-fold genome coverage, trypanosomes were induced to express double-stranded RNA and screened for reduced con canavalin A (conA) binding. Since this lectin binds the surface glycoprotein EP-procyclin, we predicted that cells would lose affinity to conA if RNAi silenced genes affecting EP-procyclin expression or modification. We found a cell line in which RNAi switches expression from glycosylated EP-procyclins to the unglycosylated GPEET-procyclin. This switch results from silencing a hexokinase gene. The relationship between procyclin expression and glycolysis was supported by silencing other genes in the glycolytic pathway, and confirmed by observation of a similar upregulation of GPEET- procyclin when parental cells were grown in medium depleted of glucose. These data suggest that T.brucei 'senses' changes in glucose level and modulates procyclin expression accordingly.
Department of Biological Chemistry, Johns Hopkins Medical School, 725 N. Wolfe Street, Baltimore, MD 21205, USA.
James C
JC
Morris
Zefeng
Z
Wang
Mark E
ME
Drew
Paul T
PT
Englund
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System; D003208:Concanavalin A / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D015723:Gene Library; D020868:Gene Silencing; D017125:Genes, Protozoan; D005947:Glucose / Q000494:pharmacology; D006019:Glycolysis / Q000235:genetics / Q000502:physiology*; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006593:Hexokinase / Q000096:biosynthesis / Q000235:genetics*; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000235:genetics*; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D011770:Pyruvate Kinase / Q000096:biosynthesis / Q000235:genetics; D012330:RNA, Double-Stranded / Q000096:biosynthesis; D016053:RNA, Protozoan / Q000235:genetics*; D034741:RNA, Small Interfering; D022661:RNA, Untranslated / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism
C406750:EP procyclin protein, Trypanosoma brucei; C406751:GPEET protein, Trypanosoma brucei; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D015800:Protozoan Proteins; D012330:RNA, Double-Stranded; D016053:RNA, Protozoan; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; D003208:Concanavalin A; D006593:Hexokinase; D011770:Pyruvate Kinase; D005947:Glucose