Functional expression of rat GLUT 1 glucose transporter in Dictyostelium discoideum

被引:17
作者
Cohen, NR
Knecht, DA
Lodish, HF
机构
[1] WHITEHEAD INST BIOMED RES,CAMBRIDGE,MA 02142
[2] UNIV CONNECTICUT,DEPT MOLEC & CELL BIOL,STORRS,CT 06269
[3] MIT,DEPT BIOL,CAMBRIDGE,MA 02139
关键词
D O I
10.1042/bj3150971
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
To facilitate expression of the rat GLUT 1 glucose transporter cDNA in Dictyostelium discoideum, we mutated the 5' end of the coding sequence such that the codons for the first ten amino acids conformed to preferred Dictyostelium codon usage. As determined by Western-blot analysis, a population of Dictyostelium transformed with the mutated cDNA expressed nonglycosylated GLUT 1 protein. Cell lines expressing GLUT transport radiolabelled 2-deoxy-D-glucose at a rate 6-10 times that of cell lines transformed with vector alone, The initial rate of inward transport of 2-deoxy-D-glucose was stimulated severalfold by the presence of unlabelled glucose in the Dictyostelium cytoplasm, exemplifying the trans-activation of GLUT 1 transport characteristic of GLUT 1 present in erythrocyte membranes. The K-m and K-i values for 2-deoxy-D-glucose, D-glucose, D-mannose and D-galactose were 3.7 mM, 2.6 mM, 11 mM and 30 mM respectively, similar to the values for GLUT 1 expressed in mammalian cells. L-Glucose and L-galactose, which are not transported by GLUT 1, do not inhibit uptake of 2-deoxy-D-glucose in Dictyostelium expressing GLUT 1. Thus, even though GLUT 1 expressed in Dictyostelium is not N-glycosylated, it transports hexoses normally; this is the first example of functional expression of a mammalian transport protein in this lower eukaryote.
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页码:971 / 975
页数:5
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