title: Expression of a novel marine viral single-chain serine palmitoyltransferase and construction of yeast and mammalian single-chain chimera.
abstract: The genus Coccolithovirus is a recently discovered group of viruses that infect the globally important marine calcifying microalga Emiliania huxleyi. Surprisingly, the viral genome contains a cluster of putative sphingolipid biosynthetic genes not found in other viral genus. To address the role of these genes in viral pathogenesis, the ehv050 gene predicted to encode a serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of sphingolipid biosynthesis, was expressed and characterized in Saccharomyces cerevisiae. We show that the encoded protein is indeed a fully functional, endoplasmic reticulum-localized, single-chain SPT. In eukaryotes SPT is a heterodimer comprised of long chain base 1 (LCB1) and LCB2 subunits. Sequence alignment and mutational analysis showed that the N-terminal domain of the viral protein most closely resembled the LCB2 subunit and the C-terminal domain most closely resembled the LCB1 subunit. Regardless of whether the viral protein was expressed as a single polypeptide or as two independent domains, it exhibited an unusual preference for myristoyl-CoA rather than palmitoyl-CoA. This preference was reflected by the increased presence of C16-sphingoid bases in yeast cells expressing the viral protein. The occurrence of a single-chain SPT suggested to us that it might be possible to create other fusion SPTs with unique properties. Remarkably, when the two subunits of the yeast SPT were thus expressed, the single-chain chimera was functional and displayed a novel substrate preference. This suggests that expression of other multisubunit membrane proteins as single-chain chimera could provide a powerful approach to the characterization of integral membrane proteins.
authors.affiliation: Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20184, USA.
authors.forename: Gongshe
authors.initials: G
authors.lastname: Han
authors.forename: Kenneth
authors.initials: K
authors.lastname: Gable
authors.forename: Lianying
authors.initials: L
authors.lastname: Yan
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Allen
authors.forename: William H
authors.initials: WH
authors.lastname: Wilson
authors.forename: Prasun
authors.initials: P
authors.lastname: Moitra
authors.forename: Jeffrey M
authors.initials: JM
authors.lastname: Harmon
authors.forename: Teresa M
authors.initials: TM
authors.lastname: Dunn
mesh_terms: D000595:Amino Acid Sequence; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000235:genetics*; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000138:chemical synthesis / Q000235:genetics; D008969:Molecular Sequence Data; D017862:Phycodnaviridae / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D021122:Protein Subunits / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D051102:Serine C-Palmitoyltransferase / Q000096:biosynthesis / Q000138:chemical synthesis / Q000235:genetics*; D014764:Viral Proteins / Q000096:biosynthesis / Q000138:chemical synthesis / Q000235:genetics
chemical_list: D004277:DNA, Single-Stranded; D008565:Membrane Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014764:Viral Proteins; C495087:LCB2 protein, S cerevisiae; C495089:SPTLC2 protein, human; D051102:Serine C-Palmitoyltransferase