Fig. 1.

The exoplasmic loops of SMSr and SMS1 harbor structural determinants of substrate selectivity. A: Predicted membrane topology of SMSr and SMS1. Active site residues are marked in red. The positions of two N-linked glycosylation sites introduced by site-directed mutagenesis in human SMSr are indicated. SAM, sterile α motif. B: Anti-V5 immunoprecipitates prepared from HeLa cells transfected with V5-tagged human SMSr, SMSr^M180N, or SMSr^G284N constructs were mock-treated or treated with EndoH and then subjected to immunoblot analysis with anti-V5 antibody. SMSr-G marks the migration of an N-glycosylated EndoH-sensitive form of SMSr that is produced exclusively in SMSr^M180N- and SMSr^G284N-expressing cells. C: Sequence alignment of loop c in SMS family members from vertebrates and fruit fly. The position of a residue critical for discriminating the phospholipid head group donors, PC and PE (Asp or Glu), is marked by an arrow. Database accession numbers are: human SMS1, BAD16809.1; chicken SMS1, ADY69193.1; frog SMS1, NP_001008197.1; zebrafish SMS1, NP_001071082.1; human SMS2, NP_689834.1; chicken SMS2, XP_420492.1; frog SMS2, AAH88568.1; zebrafish SMS2, zgc:100911; human SMSr, Q96LT4; chicken SMSr, XP_426501.3; frog SMSr, Q28CJ3; zebrafish SMSr, zgc:162183; fruit fly SMSr, CG32380. D: TLC analysis of reaction products formed when lysates of yeast strains expressing V5-tagged human SMS2 or SMS2^E271D were incubated with C₆-NBD-ceramide (NBD-Cer). EV denotes yeast lysate from strain transfected with empty vector. SMS expression was verified by immunoblotting with anti-V5 antibody (bottom). Note that residue substitution E271D in loop c converts SMS2 into a monofunctional SMS. E: TLC analysis of reaction products formed when lysates of yeast strains expressing V5-tagged human SMSr, SMSr^E343D, SMS1, SMS1^D327E, or SMS1/r chimera in which loop b was swapped (SMSr^Lb1, SMS1^Lbr) were incubated with NBD-Cer. SMS expression was verified by immunoblotting with anti-V5 antibody (bottom). Note that residue substitution D327E combined with swapping loop b against that of SMSr converts SMS1 into a monofunctional CPE synthase (SMS1^D327E-Lbr or SMS1^CPE). Data shown in (D) and (E) are representative of two independent experiments.