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doi:10.1016/S0166-6851(99)00093-6 
Copyright © 1999 Elsevier Science B.V. All rights reserved.
A Rab1 homologue with a novel isoprenylation signal provides insight into the secretory pathway of Theileria parva*1
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Rozmin Janoo1, Anthony Musoke, Clive Wells and Richard Bishop
, 
Received 19 February 1999;
Abstract
As a first step in developing compartment-specific markers for protein trafficking within Theileria parva, we have isolated cDNAs encoding homologues 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 that 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, including all known Rab1 homologues, in containing only a single cysteine. [C14]mevalonic acid lactone and [H3]geranylgeranyl pyrophosphate were specifically incorporated into recombinant TpRab1 in vitro, demonstrating that the novel motif was functional for isoprenylation. Recombinant TpRab1 bound radiolabeled 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 sequence as compared to the introns of other lower eukaryote Rab1 genes. Immunofluorescence microscopy using antiserum specific for the novel C-terminal peptide in combination with labelling of cells using the nucleic acid-staining dye DAPI, indicated that TpRab1 was located in the vicinity of the schizont nucleus within the infected lymphocyte.
Author Keywords: Rab GTPases; Secretory pathway; Isoprenylation; Intron; Theileria parva
Abbreviations: ATP, adenosine triphosphate; GDP, guanosine diphosphate; GTP, guanosine triphosphate; TpRab1, Theileria parva Rab1
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Amplification of Rab homologues using reverse transcription PCR
- 2.2. Nucleotide sequencing
- 2.3. Screening of cDNA libraries and Southern blot analysis
- 2.4. Expression and purification of recombinant Rab proteins in E. coli
- 2.5. Generation of antiserum against synthetic peptides, SDS polyacrylamide electrophoresis and western blot analysis
- 2.6. GTP-binding to recombinant proteins
- 2.7. Isoprenylation of Rab proteins in vitro
- 2.8. Immunofluorescence microscopy
- 3. Results
- 3.1. cDNA cloning and deduced amino acid sequence of a T. parva Rab1 homologue
- 3.2. The TpRab1 gene contains four introns
- 3.3. Genomic organisation and transcription of TpRab1
- 3.4. Expression of TpRab1 and generation of polyclonal antisera against the C-terminal domain
- 3.5. GTP-binding to recombinant Rab proteins
- 3.6. Isoprenylation of TpRab1 in vitro
- 3.7. Localisation of TpRab1 within schizont-infected lymphocytes
- 4. Discussion
- Acknowledgements
- References
*1 Note: Nucleotide sequence data reported in this paper are available in the GenBank™, EMBL and DDBJ databases under the accession numbers, AF107763 (TpRab1 cDNA), and AF107762 (TpRab1 genomic DNA).
1 Present address: Department of Biology, Boston College, 315 Higgins Hall, Chestnut Hill, MA 02167, USA.
Corresponding author. Tel.: +254-2-630-743; fax: +254-2-631-499; email: r.bishop@cgiar.org
