Figure 1.
Schematic of the core GPI anchor glycan and its side chain modifications.
GPI anchors consist of a highly conserved carbohydrate sequence that is linked to the inositol head group (hexagon) of phosphatidylinositol (hexagon + LIPID). The core GPI glycan contains glucosamine (blue/white square) and three mannoses (Man I-III, green circles). A protein is linked to the third mannose via an ethanolamine phosphate. R indicates positions where modification of the core GPI structure can occur, and possible R modifications in various species are described in Table 1. The red R1 indicates the position of fourth mannose addition.
Table 1.
Modifications of the GPI anchor core structure observed in various organisms1.
Figure 2.
Complementation of yeast gpi10 and smp3 null mutants by protozoan PIGB genes.
A. Schematic of yeast plasmid shuffling experiments. Haploid gpi10::KanR containing pGAL-ScGPI10 (top row) or smp3::KanR containing pGAL-ScSMP3 (bottom row) strains were transformed with protozoan pPGK-PIGB expression plasmids. Yeasts carrying both plasmids were then induced to lose one plasmid via growth on rich medium, and then strains containing only the pPGK plasmids were selected for by growth on 5FOA. B. Serial drop cultures of yeast on SGal and SD + 5FOA media. Note that all strains grow on SGal medium as they contain both the pGAL rescuing plasmid and the pPGK expression plasmid. Following selection on 5FOA only the strains containing PIGB genes that can complement the indicated null mutation are able to grow.
Figure 3.
PCR confirmation of yeast strains.
A–C. Schematics of PCR reactions used to confirm strains. See Table S3 for sequences of the indicated primers. A. To confirm the presence (on SGal) or absence (on 5FOA) of the pGAL rescuing plasmid, the URA3 locus within this plasmid was amplified. B. To confirm that the endogenous gpi10 or smp3 locus was indeed null in all strains, forward primers residing in the 5′UTR of the genes were coupled with a reverse primer that sits within the kanamycin cassette that was inserted into the gene locus upon null strain generation. C. To confirm the presence of the pPGK plasmid, PCR was performed using a primer within the pPGK vector, coupled with a primer in the 3′end of the coding region of the PIGB gene of interest. D–E. Results of PCR experiments to confirm all yeast strains shown in Figure 2. DNA was isolated from all strains and the PCRs described in A–C were performed. D. gpi10Δ strains before (SGal) and after (5FOA) plasmid shuffling. E. smp3Δ strains before (SGal) and after (5FOA) plasmid shuffling. Reactions A–C correspond to the schematics A–C at left. Amplicon sizes are: Reaction A: 500 bp; Reaction B: 534 bp for gpi10, 616 bp for smp3; Reaction C: 902 bp for pPGK-415, 891 bp for pPGK-ScGPI10, 922 bp for pPGK-ScSMP3, 800 bp for pPGK-TbPIGB, 820 bp for pPGK-TcrPIGB, 879 bp for pPGK-TvPIGB, 932 bp for pPGK-TcoPIGB and 908 bp for pPGK-PfPIGB.
Figure 4.
T. cruzi PIGB can complement a yeast strain deficient in both gpi10 and smp3.
A. Schematic of a tetrad showing the desired non-parental ditype pattern of kanamycin marker segregation generated from the mating of a haploid gpi10::KanR strain containing pGAL-TcrPIGB with a haploid smp3::KanR strain containing pGAL-TcrPIGB. B. PCR was used to test each haploid meiotic segregant from a non-parental ditype tetrad for the presence of gpi10Δ and smp3Δ alleles (see Figure 3B for PCR schematic). Haploids 1 and 2 are each double mutants that contain both null alleles. C. Serial drop cultures of both single gpi10Δ and smp3Δ mutant strains as well as the double mutant gpi10Δ/smp3Δ strain. Note that the strains grow on the galactose-containing medium that induces pGAL-TcrPIGB expression but not on the glucose medium, which represses pGAL-TcrPIGB expression.
Figure 5.
The long and short conserved motifs do not confer broad specificity to TcrPigB.
A. Alignment of the N-terminal long and C-terminal short motifs from yeast and protozoa. B. Schematics of the generated chimera constructs. The N-terminal long (chimera 1), C-terminal short (chimera 2) or both conserved motifs (chimera 3) of TcrPIGB (red) were replaced by those from TbPIGB (green). Numbers correspond to the amino acid positions of each domain within its native protein sequence. C. Serial drop cultures of control and chimeric constructs in both gpi10Δ and smp3Δ strains before (SGal) and after (SD + 5FOA) plasmid shuffling. Note that all three of the chimeric PigB proteins are able to complement the gpi10Δ and smp3Δ mutant strains.