Functional expression of the murine Golgi CMP-sialic acid transporter in Saccharomyces cerevisiae

Abstract

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 K(m) of 2.9 μM. 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.