Interactions of y+LAT1 and 4F2hc in the y+l amino acid transporter complex: consequences of lysinuric protein intolerance-causing mutations.
Lysinuric protein intolerance (LPI) is an inherited aminoaciduria caused by recessive mutations in the SLC7A7 gene encoding y+L amino acid transporter 1 (y+LAT1), which combines with 4F2hc to generate an active transporter responsible for the system y+L amino acid transport. We have previously shown that the y+LAT1 proteins with point mutations are expressed in the plasma membrane, while those with frameshift mutations are retained in the cytoplasm. This finding has prompted us to study whether the difference in localization is due to the inability of the structurally altered mutant y+LAT1 proteins to heteromerize with 4F2hc. For this purpose, we utilized FACS technique to reveal fluorescence resonance energy transfer (FRET) in cells expressing wild type or LPI-mutant CFP-tagged y+LAT1 and YFP-tagged 4F2hc. The heteromerization of y+LAT1 and 4F2hc within the cell is not disrupted by any of the tested LPI mutations. In addition, the expression rate of the LPI mutant y+LAT1 proteins was significantly lower and cellular mortality was markedly increased than that of the wild type y+LAT1 in transfected samples. Our results indicate that the FACS-FRET method provides an alternative approach for screening of potential protein associations.
Department of Medical Biochemistry and Genetics, Institute of Biomedicine, University of Turku, Turku, Finland. minna.toivonen@utu.fi.
Minna
M
Toivonen
Maaria
M
Tringham
Johanna
J
Kurko
Perttu
P
Terho
Olli
O
Simell
Kaisa M
KM
Heiskanen
Juha
J
Mykkänen
D000592:Amino Acid Metabolism, Inborn Errors / Q000235:genetics*; D027241:Amino Acid Transport System y+L; D049109:Cell Proliferation; D002470:Cell Survival / Q000235:genetics; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D027301:Fusion Regulatory Protein 1, Light Chains / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D011485:Protein Binding / Q000235:genetics; D055503:Protein Multimerization / Q000235:genetics*; D020836:Protein Structure, Quaternary
D027241:Amino Acid Transport System y+L; D027282:Fusion Regulatory Protein 1, Heavy Chain; D027301:Fusion Regulatory Protein 1, Light Chains; C545989:SLC3A2 protein, human; C421261:SLC7A7 protein, human