FIGURE 4:

trappc11 mutants have glycosylation defects. (A) Livers and liverless carcasses from Tg(fabp10:Gc-EGFP) WT and trappc11 mutants at 5 dpf were collected and analyzed by Western blot for glycosylation of Gc-EGFP, with β-actin serving as a loading control. Blotting Gc-EGFP from WT whole-embryo lysates subjected to PNGase F digestion (+) serves as a control for hypoglycosylated Gc-EGFP. (B) LLO from 5-dpf WT and trappc11 mutants, and WT larvae treated with 1 μg/ml Tm or DMSO, were purified and compared to DMSO-treated larvae analyzed by FACE. Standard marker separation (std) for size comparison is shown. (C) Amounts of full-length (Glc₃Man₉) LLO from B were quantified by ImageJ (National Institutes of Health, Bethesda, MD) densitometric analysis. trappc11 mutants have ∼25% LLO compared with WT siblings (n = 5). Tm treatment of WT embryos also results in reduced LLO levels (n = 2). (D) FACE analysis of N-linked glycans removed from proteins demonstrates that trappc11 mutants (n = 5) have reduced glycosylation of proteins, also consistent with that seen in larvae treated with 1 μg/ml Tm (n = 2). Values in trappc11 mutants were normalized to phenotypically WT siblings, and Tm-treated WT larvae were normalized to DMSO-treated controls. (E) Livers from WT siblings and trappc11 mutants were collected and analyzed at 5 dpf for the expression of genes involved in N-linked glycosylation. Fold increases in expression was compared with WT siblings (for trappc11) or with DMSO-treated WT larvae for Tm and BFA-treated larvae (Tm and BFA-treated). There was significant up-regulation of genes involved in glycosylation in trappc11 mutants (Student’s t test). A similar increase is found in larvae treated with 1 μg/ml Tm, but not in those treated with 1 μg/ml BFA from 3 to 5 dpf. n.s., not significant. (F) Activity assays for Mpi and Pmm2 in 5 dpf WT and trappc11-mutant larvae. Tm-treated larvae (1 μg/ml) show comparable increase in Pmm2, but Mpi activity was not changed in trappc11 in mutants or Tm-treated larvae. These changes are consistent with the gene expression shown in E. Mean ± SD of three independent clutches. The p values were calculated using Student’s t test; n.s., not significant.