Berninsone P, Eckhardt M, Gerardy-Schahn R, Hirschberg C B
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655, USA.
J Biol Chem. 1997 May 9;272(19):12616-9. doi: 10.1074/jbc.272.19.12616.
We have functionally expressed the murine Golgi putative CMP-sialic acid transporter in Saccharomyces cerevisiae. Using a galactose-inducible expression system, S. cerevisiae vesicles were able to transport CMP-sialic acid. Transport was dependent on galactose induction and was temperature-dependent and saturable with an apparent Km of 2.9 microM. Transport was inhibited by CMP, and upon vesicle disruption with Triton X-100 parameters were very similar to the previously described CMP-sialic acid transport characteristics observed with mammalian Golgi vesicles. CMP-sialic acid transport induction was specific as no transport of UDP-galactose was observed even though the latter putative transporter has a high degree of amino acid sequence identity with the CMP-sialic acid transporter. Together, the above results demonstrate that the previously described cDNA encoding the putative CMP-sialic acid transporter encodes the transporter protein per se and suggests that this heterologous expression system may be used for further structural and functional studies of other Golgi membrane transporter proteins.
我们已在酿酒酵母中功能性表达了小鼠高尔基体假定的CMP-唾液酸转运蛋白。利用半乳糖诱导表达系统，酿酒酵母囊泡能够转运CMP-唾液酸。转运依赖于半乳糖诱导，具有温度依赖性且可饱和，表观Km为2.9微摩尔。转运受到CMP的抑制，用 Triton X-100破坏囊泡后，其参数与先前在哺乳动物高尔基体囊泡中观察到的CMP-唾液酸转运特征非常相似。CMP-唾液酸转运诱导具有特异性，因为即使后者假定的转运蛋白与CMP-唾液酸转运蛋白具有高度的氨基酸序列同一性，也未观察到UDP-半乳糖的转运。综上所述，上述结果表明，先前描述的编码假定CMP-唾液酸转运蛋白的cDNA本身编码转运蛋白，并表明这种异源表达系统可用于进一步研究其他高尔基体膜转运蛋白的结构和功能。
