Cloning and identification of Rab cDNAs from Limulus polyphemus

Small GTPases of the Rab family are essential for the control of membrane transport between intracellular compartments. Trafficking of the sodium-dependent facilitative insulin responsive glucose transporter (GLUT 4) has been shown to be associated with the intracellular redistribution of Rabs 4, 5 and 11 in adipose and muscle tissues. As a prelude to studies of the endosomal trafficking of the choline cotransporter (ChCoT), we describe herein our initial efforts to identify Rab proteins in Limulus polyphemus central nervous system (CNS) tissue. The studies were initiated after results from Microarray analysis of Limulus RNA hybridized to mouse gene chips suggested the presence of RNA transcripts for Rab 7 protein. Subsequently, more than 30 sequences for different Rab proteins were aligned and several consensus segments were selected for degenerate primer design to produce Rabs 2, 4, 7, 9 and 11. The expected PCR fragment sizes were obtained using RT-PCR and subcloned into pCR II TOPO vector and transferred into E. coli Top 10. The nucleotide sequences indicated that the recombinants encoded partial amino acid sequences for Rabs 1a, 1b, 1c, 2, 2a, 2b, 3a, 4, 5a, 7a, 7b, 11a, 11b, 14, 33b1 and 33b2. Northern blot analyses showed that the molecular sizes of Limulus Rabs 3a, 4, 7, 11a and 11b ranged from approximately 1.94.6 Kb. These Rab proteins, particularly Rabs 4, 7 and 11, will be studied further to determine their possible roles in the trafficking of the Limulus ChCoT