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. 2018 Jul 17;7:e38752. doi: 10.7554/eLife.38752

Figure 1. BiP is a target of Fic AMPylation and deAMPylation in vivo.

(A) BiP AMPylation during times of low ER stress reserves a portion of the chaperone to allow for a rapid, deAMPylation-driven, response to high ER stress (Casey et al., 2017; Preissler et al., 2017a). (B) Bar graphs show the percentage of null mutant BiPG0102/y males rescued by the indicated genomic BiPWT, BiPT366A or BiPT518A genomic transgene (Tg) relative to sibling controls. N = 3 biological replicas. At least 50 flies scored for each replica. Bar graphs show means ± Standard Deviation (SD). (C) Bar graphs show the percentage of viable flies of the indicated wild-type or fic30C genotypes expressing the overactive FicE247G under the ubiquitous Da-Gal4 driver relative to sibling controls. Among the indicated genomic BiP transgenes, only BiPT366A provides partial rescue of lethality in the BiP+/+ background and near complete rescue in a BiPG0102 null background. N = 3 biological replicas. At least 100 total flies scored for each replica. Bar graphs show means ± SD.

Figure 1—source data 1. Relates to Figure 1B and C.
Quantification of fly survival.
elife-38752-fig1-data1.xlsx (31.4KB, xlsx)
DOI: 10.7554/eLife.38752.006

Figure 1.

Figure 1—figure supplement 1. Expression of genomic BiP transgenes.

Figure 1—figure supplement 1.

(A) Western blots for FLAG-tagged BiP transgenes and total BiP in whole head lysates in BiP wild type or homozygous mutant background as indicated. Actin (JLA-20) served as a loading control. (B) Kar2T538A mutants have temperature-sensitive growth defects. Yeast strains kar2Δ + pKar2, kar2Δ + pkar2T386A, and kar2Δ + pkar2T538A were grown at 25°C and five-fold serially diluted onto plates of rich media incubated at the indicated temperatures.

Figure 1—figure supplement 2. AMPylation-resistant BiPT366A phenocopies fic.

Figure 1—figure supplement 2.

(A) ERGs of fic30C flies show reduced ON transients (arrows). Flies homozygous for a lethal BiPG0102 allele rescued by BiPWT transgene have normal vision but flies rescued with the mutant BiPT366A transgene display reduced ON transients. (B) Quantification of ERG traces. Bar graphs show means ± SD. ****p<0.0001; ***p<0.001; *p<0.05; n = 24 flies per genotype and condition.
Figure 1—figure supplement 2—source data 1. Relates to Figure 1—figure supplement 2B.
Quantification of ERG components (white-).
elife-38752-fig1-figsupp2-data1.xlsx (45.1KB, xlsx)
DOI: 10.7554/eLife.38752.007

Figure 1—figure supplement 3. Red eye pigment suppresses ERG phenotypes of fic30C and BiPT366A mutants.

Figure 1—figure supplement 3.

(A) ERGs of OreR and red-eyed fic30C flies as well as BiPWT and BiPT366A animals. (B) Quantification of ERG data. Bar graphs show means ± SD. **p<0.01, n = 18 flies per genotype and condition.
Figure 1—figure supplement 3—source data 2. Relates to Figure 1—figure supplement 3B.
Quantification of ERG components (white+).
elife-38752-fig1-figsupp3-data2.xlsx (42.2KB, xlsx)
DOI: 10.7554/eLife.38752.008