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 re gulate the flux through individual steps of the intracellular membrane traf ficking pathway, such as ER-to-Golgi transport, probably by controlling SNA RE complex assembly. In Trypanosoma brucei a number of Rab proteins have be en isolated by EST analysis; here we characterise one of these, TbRab2p (or iginally designated Trab1p), which is a member of the Ypt1p subfamily of Ra b proteins. Recombinant TbRab2p is capable of hydrolysing GTP and is post-t ranslationally modified in vitro by addition of a geranylgeranyl prenyl gro up, properties of an authentic Rab GTPase, Antibodies against recombinant T bRab2p show that in trypanosomes TbRab2p is localised primarily to the endo plasmic reticulum (ER) and colocalises with BiP in wild-type trypanosomes, Over expression of TbRab2p in procyclic form T. brucei results in a cell po pulation having a 40-fold increase in TbRab2p expression. In these cells bi osynthesis of procyclin, a secretory pathway glycoprotein, is decreased, ac companied by an increase in general protein biosynthesis, suggesting that e xcess TbRab2p affects ER function. Heterologous expression of TbRab2p in CO S cells resulted in targeting to the pre-Golgi transport intermediate (ERGI C), indicating that the targeting information is conserved between mammals and trypanosomes, Clustal and phylogenetic analyses support assignment of T bRab2p as a Rab2 homologue, In addition, over expression of TbRab2p in tryp anosomes results in membrane reorganisation and formation of opaque vesicul ar structures visible by phase contrast microscopy, consistent with accumul ation of ER-derived vesicular structures in cells highly overexpressing TbR ab2p, Ultrastructural examination by electron microscopy confirmed the pres ence of a tubulo-vesicular membrane bound compartment in close proximity to the cis-Golgi, probably equivalent to the ERGIC, TbRab2p is therefore a ne w ER/ERGIC marker for T. brucei.