Expression and activity of chimeric molecules between human UDP-galactose transporter and CMP-sialic acid transporter.
Human UDP-galactose transporter (hUGT1) and CMP-sialic acid transporter (hCST) are related Golgi proteins with eight putative transmembrane helices predicted by computer analysis. We constructed chimeric molecules in which segments of various lengths from the C- or N-terminus of hUGT1 were replaced by corresponding portions of hCST. The chimeras were transiently expressed in UGT-deficient mutant Lec8 cells, and their UGT activity was assessed by the binding of GS-II lectin to the transfected cells. The replacement of either the N- or C-terminal cytoplasmic segment by that of hCST did not affect the expression or activity of hUGT1. A chimera in which the eighth helix and the C-terminal tail were replaced also retained the UGT activity, indicating that this helix is not involved in the determination of substrate specificity. In contrast, three types of chimeras, in which the first helix, the first and the second helices, and a segment from the seventh helix to the C-terminus were replaced, respectively, were expressed very infrequently in the transfected cells, and had no UGT activity. They are likely folded incorrectly and degraded by a quality-control system, since the amounts of their mRNAs were normal and the proteins were mainly localized in the ER. The first and the seventh helices are important for the stability of the transporter protein.
Aoki
K
K
Department of Physiological Chemistry, The Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo, 113-8613, Japan.
Sun-Wada
G H
GH
Segawa
H
H
Yoshioka
S
S
Ishida
N
N
Kawakita
M
M
D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D016466:CHO Cells; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006224:Cricetinae; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009154:Mutation; D033704:Nucleotide Transport Proteins; D017433:Protein Structure, Secondary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014531:Uridine Diphosphate Galactose / Q000378:metabolism; D000099237:UDP-Galactose Translocators
D002352:Carrier Proteins; D003569:Cytidine Monophosphate N-Acetylneuraminic Acid; D017931:DNA Primers; D004274:DNA, Recombinant; D008565:Membrane Proteins; D009004:Monosaccharide Transport Proteins; D033704:Nucleotide Transport Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014531:Uridine Diphosphate Galactose; D000099237:UDP-Galactose Translocators; C491081:SLC35A1 protein, human