Identification of TbPBN1 in Trypanosoma brucei reveals a conserved heterodimeric architecture for glycosylphosphatidylinositol‐mannosyltransferase‐I

Glycosylphosphatidylinositol (GPI)‐anchored proteins are found in all eukaryotes and are especially abundant on the surface of protozoan parasites such as Trypanosoma brucei. GPI‐mannosyltransferase‐I (GPI‐MT‐I) catalyzes the addition of the first of three mannoses that make up the glycan core of GPI. Mammalian and yeast GPI‐MT‐I consist of two essential subunits, the catalytic subunit PIG‐M/Gpi14 and the accessory subunit PIG‐X/Pbn1(mammals/yeast). T. brucei GPI‐MT‐I has been highlighted as a potential antitrypanosome drug target but has not been fully characterized. Here, we show that T. brucei GPI‐MT‐I also has two subunits, TbGPI14 and TbPBN1. Using TbGPI14 deletion, and TbPBN1 RNAi‐mediated depletion, we show that both proteins are essential for the mannosyltransferase activity needed for GPI synthesis and surface expression of GPI‐anchored proteins. In addition, using native PAGE and co‐immunoprecipitation analyses, we demonstrate that TbGPI14 and TbPBN1 interact to form a higher‐order complex. Finally, we show that yeast Gpi14 does not restore GPI‐MT‐I function in TbGPI14 knockout trypanosomes, consistent with previously demonstrated species specificity within GPI‐MT‐I subunit associations. The identification of an essential trypanosome GPI‐MT‐I subcomponent indicates wide conservation of the heterodimeric architecture unusual for a glycosyltransferase, leaving open the question of the role of the noncatalytic TbPBN1 subunit in GPI‐MT‐I function.