The topology of the Lcb1p subunit of yeast serine palmitoyltransferase.
The structural organization and topology of the Lcb1p subunit of yeast and mammalian serine palmitoyltransferases (SPT) were investigated. In the yeast protein, three membrane-spanning domains were identified by insertion of glycosylation and factor Xa cleavage sites at various positions. The first domain of the yeast protein, located between residues 50 and 84, was not required for the stability, membrane association, interaction with Lcb2p, or enzymatic activity. Deletion of the comparable domain of the mammalian protein SPTLC1 also had little effect on its function, demonstrating that this region is not required for membrane localization or heterodimerization with SPTLC2. The second and third membrane-spanning domains of yeast Lcb1p, located between residues 342 and 371 and residues 425 and 457, respectively, create a luminal loop of approximately 60 residues. In contrast to the first membrane-spanning domain, the second and third membrane-spanning domains were both required for Lcb1p stability. In addition, mutations in the luminal loop destabilized the SPT heterodimer indicating that this region of the protein is important for SPT structure and function. Mutations in the extreme carboxyl-terminal region of Lcb1p also disrupted heterodimer formation. Taken together, these data suggest that in contrast to other members of the alpha-oxoamine synthases that are soluble homodimers, the Lcb1p and Lcb2p subunits of the SPT heterodimer may interact in the cytosol, as well as within the membrane and/or the lumen of the endoplasmic reticulum.
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD 20184-4799, USA.
Gongshe
G
Han
Ken
K
Gable
Lianying
L
Yan
Mukil
M
Natarajan
Jayasree
J
Krishnamurthy
Sita D
SD
Gupta
Anna
A
Borovitskaya
Jeffrey M
JM
Harmon
Teresa M
TM
Dunn
D000217:Acyltransferases / Q000737:chemistry* / Q000378:metabolism; D000483:Alleles; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D003062:Codon; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D004721:Endoplasmic Reticulum / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry; D017353:Gene Deletion; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D051102:Serine C-Palmitoyltransferase
D003062:Codon; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000217:Acyltransferases; C487784:SPTLC1 protein, human; D051102:Serine C-Palmitoyltransferase; D015951:Factor Xa