Localization, topology, and function of the LCB1 subunit of serine palmitoyltransferase in mammalian cells.
Serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the biosynthesis of sphingolipids, comprises two different subunits, LCB1 and LCB2. LCB1 has a single highly hydrophobic domain near the N terminus. Chinese hamster ovary cell mutant LY-B cells are defective in SPT activity because of the lack of expression of an endogenous LCB1 subunit. Stable expression of LCB1 having an epitope tag at either the N or C terminus restored SPT activity of LY-B cells, suggesting that the epitope tag did not affect the localization or topology of LCB1. Indirect immunostaining showed that the N- and C-terminal epitopes are oriented toward the lumenal and cytosol side, respectively, at the endoplasmic reticulum. Interestingly, there was far less LCB2 in LY-B cells than in wild-type cells, and the amount of LCB2 in LY-B cells was restored to the wild-type level by transfection with LCB1 cDNA. In addition, overproduction of the LCB2 subunit required co-overproduction of the LCB1 subunit. These results indicated that the LCB1 subunit is most likely an integral protein having a single transmembrane domain with a lumenal orientation of its N terminus in the endoplasmic reticulum and that the LCB1 subunit is indispensable for the maintenance of the LCB2 subunit in mammalian cells.
Yasuda
Satoshi
S
Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
Nishijima
Masahiro
M
Hanada
Kentaro
K
D000217:Acyltransferases / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D000939:Epitopes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D011233:Precipitin Tests; D051102:Serine C-Palmitoyltransferase
D017931:DNA Primers; D000939:Epitopes; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase