Functional consequences of the in-frame insertion of a transposon into the mutated gamma amino butyric acid transporter of Saccharomyces cerevisiae.
BACKGROUND
The Saccharomyces cerevisiae strain, 22574d, lacks gamma amino butyric acid (GABA) transport activity and cannot grow on this amino acid as sole nitrogen source. Both transport of and growth on this amino acid are restored when the yeast is transformed with a form of mouse gamma actin containing an extended C-terminal sequence (M-g-A). The nature of the mutation in the transporter as well as the complementation mode are addressed.


METHODS
The cDNA sequence of the mutated transporter was achieved using reverse transcription, 3'-Race and cloning. For detection, Northern blotting and labeling with 32-P were used. The putative ability of the transporter to interact with gamma actin in vivo was examined by following the interaction in vitro of synthetic peptides corresponding to the C-termini of the gamma actin and the transporter.


RESULTS
Up to codon 394 the mutated and native transporters are identical. At the 3' end, the mutant is by extended by 32 codons from the delta region of a Ty1 transposon, giving an open reading frame of 426 codons, and a predicted structure of 9 of the 11 transmembrane domains. Peptides corresponding to the carboxy terminal regions of the truncated transporter and the elongated species of gamma actin show the potential to interact in vitro.


CONCLUSIONS
The mutated GABA transporter mRNA contains an insert from the delta region of a Ty1 transposon. This insertion allows expression of a transporter capable of interacting with elongated gamma actin to rehabilitate the transporter.
Naghibalhossaini
Fakhraddin
F
Department of Biochemistry, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran.
Nault
Francine
F
Saragovi
Uri
U
Nedev
Haynu
H
Johnstone
Rose
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D003062:Codon; D004251:DNA Transposable Elements; D018076:DNA, Complementary / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D004996:Ethidium / Q000494:pharmacology; D005656:Fungal Proteins / Q000378:metabolism; D050485:GABA Plasma Membrane Transport Proteins; D007364:Intercalating Agents / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D027361:Organic Anion Transporters / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors
D002352:Carrier Proteins; D003062:Codon; D004251:DNA Transposable Elements; D018076:DNA, Complementary; D005656:Fungal Proteins; D050485:GABA Plasma Membrane Transport Proteins; D007364:Intercalating Agents; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D027361:Organic Anion Transporters; D010455:Peptides; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; C513599:UGA4 protein, S cerevisiae; D012313:RNA; D004996:Ethidium