Molecular cloning and functional expression of a proline transporter from Manduca sexta.
We report the molecular cloning of a L-proline transporter, MasPROT cDNA and its splice variants MasPROT.16 and MasPROT.2 from the central nervous system of Manduca sexta. Sequence analysis revealed that MasPROT belongs to a family of high affinity Na+/Cl- dependent neurotransmitter transporters. The deduced amino acid (aa) sequence of 556 aa having an estimated molecular mass of 58.9 kDa is predicted to have 12 putative transmembrane domains (TMD) and a characteristic large extracellular loop between TMD3 and TMD4. Sequence comparison to other members of the family indicates that it falls into the glycine-proline transporter subfamily. Transiently expressed MasPROT cDNA in Xenopus oocytes exclusively transported proline. Northern analysis shows that it is expressed predominantly in central nervous system, however, low levels are present in midgut, hindgut and Malpighian tubules. Two mRNA transcripts of sizes 3.6 and 8 Kb were found in all tissues except hindgut, where only a smaller transcript exists. RT-PCR and Southern blot analysis revealed the presence of MasPROT transcripts in flight muscles but not in leg muscles. Our preliminary data suggests that this transporter is an insect homologue of mammalian proline transporters. MasPROT.16 is a short splice variant encoding for 174 amino acids and shares 138 amino acids from the N terminus of MasPROT. MasPROT.2 is a long splice variant that contains six introns that coincide precisely with the previously mapped exon/intron boundaries of the members of this superfamily.
Sandhu
Sumandeep K
SK
Environmental Toxicology Graduate Program, University of California, Riverside CA 92521, USA.
Ross
Linda S
LS
Gill
Sarjeet S
SS
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D026921:Amino Acid Transport Systems, Neutral / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D014644:Genetic Variation; D007438:Introns; D018402:Manduca / Q000145:classification / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D014176:Protein Biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D026921:Amino Acid Transport Systems, Neutral; D017931:DNA Primers; D018076:DNA, Complementary; C033082:proline transporter