A Rab1 homologue with a novel isoprenylation signal provides insight into the secretory pathway of Theileria parva

As a first step in developing compartment-specific markers for protein traf ficking within Theileria ia parva, we have isolated cDNAs encoding homologu es of the small GTP binding proteins Rab1 and Rab4. The T. parva homologue of Rab1 (TpRab1), a protein which regulates vesicular transport between the endoplasmic reticulum and cis golgi in other organisms, was unusual in tha t it contained a unique 17 amino acid C-terminal extension. The C-terminal motif sequence KCT (XCX) contrasted with the CXC or XCC motifs which act as as signals for isoprenylation by geranylgeranyl in most Rab proteins, incl uding all known Rab1 homologues, in containing only a single cysteine. [C-1 4]mevalonic acid lactone and [H-3]geranylgeranyl pyrophosphate were specifi cally incorporated into recombinant TpRab1 in vitro, demonstrating that the novel motif was functional for isoprenylation. Recombinant TpRab1 bound ra diolabeled GTP, and this binding was inhibited by excess unlabeled GTP and GDP and also partially by ATP. The TpRab1 gene contained four short (34-67 bp) introns with a distinct pattern of occurrence within the protein sequen ce as compared to the introns of other lower eukaryote Rab1 genes. Immunofl uorescence microscopy using antiserum specific for the novel C-terminal pep tide in combination with labelling of cells using the nucleic acid-staining dye DAPI, indicated that TpRab1 was located in the vicinity of the schizon t nucleus within the infected lymphocyte. (C) 1999 Elsevier Science B.V. Al l rights reserved.