Functional expression of human golgi CMP-sialic acid transporter in the Golgi complex of a transporter-deficient Chinese hamster ovary cell mutant.
We recently described the cloning of putative human CMP-sialic acid transporter (hCST) cDNA [Ishida, N. et al. (1996) J. Biochem. 120, 1074-1078]. The hCST cDNA coded for a hydrophobic protein with an amino acid sequence showing a high degree of similarity (92% identity) to that of murine CMP-sialic acid transporter. In this report, we demonstrate that hCST corrects the CMP-sialic acid transporter-deficient phenotype of CHO-derived Lec2 cells, as judged from the recovery of WGA-sensitivity by transformants, and the recovery of CMP-sialic acid transporting ability by microsomal vesicles prepared from them. A peptide antibody against the C-terminus of the hCST protein detected the cDNA products expressed in the microsomes of the transformants. The subcellular localization of the hCST protein in the Golgi membrane was demonstrated by immunofluorescence microscopy, using the hCST-specific antibody. These results clearly indicate that hCST cDNA encodes the human CMP-sialic acid transporter protein. Plasma membrane-selective permeabilization combined with immunofluorescence microscopy provided strong evidence that the C-terminus of the human CMP-Sia transporter is exposed to the cytosol on the outer surface of the Golgi membrane.
Ishida
N
N
Department of Physiological Chemistry, The Tokyo Metropolitan Institute of Medical Science (Rinshoken), Bunkyo-ku, Tokyo, 113-8613, Japan.
Ito
M
M
Yoshioka
S
S
Sun-Wada
G H
GH
Kawakita
M
M
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D017931:DNA Primers; D018076:DNA, Complementary; D005822:Genetic Vectors; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D033704:Nucleotide Transport Proteins; D013347:Subcellular Fractions / Q000378:metabolism; D014909:Wheat Germ Agglutinins
D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D008565:Membrane Proteins; D033704:Nucleotide Transport Proteins; C491081:SLC35A1 protein, human; D014909:Wheat Germ Agglutinins