title: Localization, topology, and function of the LCB1 subunit of serine palmitoyltransferase in mammalian cells.
abstract: 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.
authors.affiliation: Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Yasuda
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nishijima
authors.forename: Kentaro
authors.initials: K
authors.lastname: Hanada
mesh_terms: 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
chemical_list: D017931:DNA Primers; D000939:Epitopes; D000217:Acyltransferases; D051102:Serine C-Palmitoyltransferase