Expression cloning and characterization of a cDNA encoding a novel membrane protein required for the formation of O-acetylated ganglioside: a putative acetyl-CoA transporter.
By expression cloning using COS-1 cells stably transfected with GD3-synthase (COS-1/GD3+) as a recipient cell line, we have isolated a cDNA, termed AT-1, encoding a novel protein required for the formation of O-acetylated (Ac) gangliosides. The cDNA encodes a protein with multitransmembrane spanning domains with a leucine zipper motif. It consists of 549 amino acids and has a molecular mass of 60.9 kDa. Although both O-Ac-GD3 and O-Ac-GT3 were barely detectable in recipient cells or cells transfected with the vector alone, their amount increased significantly in transfectants containing AT-1. When semi-intact cells prepared by treatment with streptolysin O were incubated with [Ac-14C]-Ac-CoA, increased incorporation of radioactivity was found in those cells transfected with AT-1 when compared with the mock transfectants. Northern blot analysis showed two major transcripts of 3.3 and 4.3 kb in all tissues examined. Immunohistochemical study with an antibody specific to the AT-1 protein suggested that it is most probably expressed in the endoplasmic reticulum membrane. Based on these results, the protein encoded by AT-1 is suggested to be an Ac-CoA transporter that is involved in the process of O-acetylation.
Kanamori
A
A
Laboratory for Cellular Glycobiology, The Institute of Physical and Chemical Research, Wako, Saitama, Japan.
Nakayama
J
J
Fukuda
M N
MN
Stallcup
W B
WB
Sasaki
K
K
Fukuda
M
M
Hirabayashi
Y
Y
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D019556:COS Cells; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005732:Gangliosides / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D018076:DNA, Complementary; D005732:Gangliosides; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D012333:RNA, Messenger; C484549:SLC33A1 protein, human