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.