TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells.
The Rab family of small GTPases is a subset of the Ras superfamily. Rabs regulate the flux through individual steps of the intracellular membrane trafficking pathway, such as ER-to-Golgi transport, probably by controlling SNARE complex assembly. In Trypanosoma brucei a number of Rab proteins have been isolated by EST analysis; here we characterise one of these, TbRab2p (originally designated Trab1p), which is a member of the Ypt1p subfamily of Rab proteins. Recombinant TbRab2p is capable of hydrolysing GTP and is post-translationally modified in vitro by addition of a geranylgeranyl prenyl group, properties of an authentic Rab GTPase. Antibodies against recombinant TbRab2p show that in trypanosomes TbRab2p is localised primarily to the endoplasmic reticulum (ER) and colocalises with BiP in wild-type trypanosomes. Over expression of TbRab2p in procyclic form T. brucei results in a cell population having a 40-fold increase in TbRab2p expression. In these cells biosynthesis of procyclin, a secretory pathway glycoprotein, is decreased, accompanied by an increase in general protein biosynthesis, suggesting that excess TbRab2p affects ER function. Heterologous expression of TbRab2p in COS cells resulted in targeting to the pre-Golgi transport intermediate (ERGIC), indicating that the targeting information is conserved between mammals and trypanosomes. Clustal and phylogenetic analyses support assignment of TbRab2p as a Rab2 homologue. In addition, over expression of TbRab2p in trypanosomes results in membrane reorganisation and formation of opaque vesicular structures visible by phase contrast microscopy, consistent with accumulation of ER-derived vesicular structures in cells highly overexpressing TbRab2p. Ultrastructural examination by electron microscopy confirmed the presence of a tubulo-vesicular membrane bound compartment in close proximity to the cis-Golgi, probably equivalent to the ERGIC. TbRab2p is therefore a new ER/ERGIC marker for T. brucei.
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Wellcome Laboratories for Molecular Parasitology, Imperial College of Science Technology and Medicine, Department of Biochemistry, Exhibition Road, London SW7 2AY, UK.
Ali
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Sherwin
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Gull
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Croft
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Field
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D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D015415:Biomarkers; D019556:COS Cells; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D017125:Genes, Protozoan; D006056:Golgi Apparatus / Q000378:metabolism* / Q000469:parasitology / Q000648:ultrastructure; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020693:rab2 GTP-Binding Protein
D015415:Biomarkers; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins; D020693:rab2 GTP-Binding Protein