Fig. 4

A putative mechanism for substrate translocation in GtrB and DPM1 function in a zebrafish model.

(a) A speculative hypothesis is that the phosphate headgroup of UndP (red) could first bind at R290, near the cytoplasmic face of the inner membrane. (b) The substrate could then diffuse along a pathway lined with conserved hydrophobic (teal) and positively charged (blue) residues. (c) Finally, the phosphate headgroup is coordinated by R122 and R200 at the acceptor site, where catalysis occurs. (d) Mutation of hydrophobic and positively charged conserved residues lining the region between JM1 and JM2 abrogates GtrB activity. Error bars are provided as s.e.m., n=3. (e) The architecture of GtrB may be shared by the DPMS complex. CDG1e mutations G152V and S248P in the conserved JM1 and JM2 linkers are represented in purple and red, respectively. (f) Phenotypes typical of dpm1 loss-of-function mutations are fully evident at 2 days post fertilization (d.p.f.) in the zebrafish embryo. Morphants show smaller head (microcephaly) and smaller eyes, kinked tail and occasional vascular defects in the tail vein (arrowhead). Scale bar, 500 µm (g) Functional analysis of DPM1 mutants was performed by injecting human DPM1 mRNA following dpm1 morpholino injection and embryos were scored as normal or affected. Human DPM1 mRNA was able to improve the morphant phenotypes (see Supplementary Fig. 8f for details) and the effect of different mutations was normalized to rescue levels. mRNA carrying the R147A or R234A mutations (equivalent to R122 and R200 in GtrB, respectively) showed almost complete loss of function. Known human missense changes (R92S, G152V and S248P) were also tested. R92S and G152V greatly abolished protein function, whereas S248P, which leads to milder human phenotypes, showed substantial residual activity. The presumed location of G152V and S248P on the juxtamembrane region is shown. R92S is expected to reside close to the active site in the GT domain. Error bars: s.e.m., **P<0.01, ***P<0.001, unpaired Student’s t-test for each mutant, n=6 (R147A), n=6 (R234A), n=3 (R92S), n=5 (G152V), n=4 (S248P). NS, not significant.