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Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

Rodriguez-Fernandez IA, Dell'Angelica EC - PLoS ONE (2015)

Bottom Line: The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family.Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively.These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

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Validation and fine mapping of the critical region responsible for the modifier effect observed for Df(3L)eygC1.Red pigments were extracted from the heads of adult g2 mutant males carrying no deletions (—) or a single copy of the indicated deficiencies, quantified as described under Materials and Methods, and expressed as percentages of the red pigment content of male flies of the wild-type (Canton-S) line. Bars represent means + SD of 6–10 biological replicates. One-way ANOVA followed by Dunnett’s test of each group versus g2 flies carrying no deletion (black bar): ***p<0.001. Shown on the left is a schematic representation of the extent of overlap between the chromosomal region deleted in the deficiency that had been identified through screening (blue) and those deleted in independent deficiencies that elicited (red) or failed to elicit (grey) a similar modifier effect on the g2 eye color phenotype. The critical genomic region responsible for the observed modifier effect is highlighted with black dashed lines, and the relative location of genes found within this region (adapted from the FlyBase database) is depicted at the bottom.
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pone.0143026.g004: Validation and fine mapping of the critical region responsible for the modifier effect observed for Df(3L)eygC1.Red pigments were extracted from the heads of adult g2 mutant males carrying no deletions (—) or a single copy of the indicated deficiencies, quantified as described under Materials and Methods, and expressed as percentages of the red pigment content of male flies of the wild-type (Canton-S) line. Bars represent means + SD of 6–10 biological replicates. One-way ANOVA followed by Dunnett’s test of each group versus g2 flies carrying no deletion (black bar): ***p<0.001. Shown on the left is a schematic representation of the extent of overlap between the chromosomal region deleted in the deficiency that had been identified through screening (blue) and those deleted in independent deficiencies that elicited (red) or failed to elicit (grey) a similar modifier effect on the g2 eye color phenotype. The critical genomic region responsible for the observed modifier effect is highlighted with black dashed lines, and the relative location of genes found within this region (adapted from the FlyBase database) is depicted at the bottom.

Mentions: In the case of Df(3L)eygC1, four independent deficiencies were available to cover the entire genomic region deleted in the original deficiency, and two of them elicited partial suppressor effects on the phenotype of g2 flies that were akin to that observed for Df(3L)eygC1 (Fig 4). Although one of these two deficiencies, Df(3L)ED4483, carried the w+mC construct as a marker, expression of this construct was deemed low on the basis of the red pigment content of flies carrying such deficiency on a White- background (Fig 3A). Through comparison of the genomic regions deleted in the deficiencies that elicited the modifier effect and those that failed to do so, a critical region comprising 17 annotated genes was defined (Fig 4, bottom box).


Identification of Atg2 and ArfGAP1 as Candidate Genetic Modifiers of the Eye Pigmentation Phenotype of Adaptor Protein-3 (AP-3) Mutants in Drosophila melanogaster.

Rodriguez-Fernandez IA, Dell'Angelica EC - PLoS ONE (2015)

Validation and fine mapping of the critical region responsible for the modifier effect observed for Df(3L)eygC1.Red pigments were extracted from the heads of adult g2 mutant males carrying no deletions (—) or a single copy of the indicated deficiencies, quantified as described under Materials and Methods, and expressed as percentages of the red pigment content of male flies of the wild-type (Canton-S) line. Bars represent means + SD of 6–10 biological replicates. One-way ANOVA followed by Dunnett’s test of each group versus g2 flies carrying no deletion (black bar): ***p<0.001. Shown on the left is a schematic representation of the extent of overlap between the chromosomal region deleted in the deficiency that had been identified through screening (blue) and those deleted in independent deficiencies that elicited (red) or failed to elicit (grey) a similar modifier effect on the g2 eye color phenotype. The critical genomic region responsible for the observed modifier effect is highlighted with black dashed lines, and the relative location of genes found within this region (adapted from the FlyBase database) is depicted at the bottom.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643998&req=5

pone.0143026.g004: Validation and fine mapping of the critical region responsible for the modifier effect observed for Df(3L)eygC1.Red pigments were extracted from the heads of adult g2 mutant males carrying no deletions (—) or a single copy of the indicated deficiencies, quantified as described under Materials and Methods, and expressed as percentages of the red pigment content of male flies of the wild-type (Canton-S) line. Bars represent means + SD of 6–10 biological replicates. One-way ANOVA followed by Dunnett’s test of each group versus g2 flies carrying no deletion (black bar): ***p<0.001. Shown on the left is a schematic representation of the extent of overlap between the chromosomal region deleted in the deficiency that had been identified through screening (blue) and those deleted in independent deficiencies that elicited (red) or failed to elicit (grey) a similar modifier effect on the g2 eye color phenotype. The critical genomic region responsible for the observed modifier effect is highlighted with black dashed lines, and the relative location of genes found within this region (adapted from the FlyBase database) is depicted at the bottom.
Mentions: In the case of Df(3L)eygC1, four independent deficiencies were available to cover the entire genomic region deleted in the original deficiency, and two of them elicited partial suppressor effects on the phenotype of g2 flies that were akin to that observed for Df(3L)eygC1 (Fig 4). Although one of these two deficiencies, Df(3L)ED4483, carried the w+mC construct as a marker, expression of this construct was deemed low on the basis of the red pigment content of flies carrying such deficiency on a White- background (Fig 3A). Through comparison of the genomic regions deleted in the deficiencies that elicited the modifier effect and those that failed to do so, a critical region comprising 17 annotated genes was defined (Fig 4, bottom box).

Bottom Line: The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family.Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively.These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Human Genetics, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
The Adaptor Protein (AP)-3 complex is an evolutionary conserved, molecular sorting device that mediates the intracellular trafficking of proteins to lysosomes and related organelles. Genetic defects in AP-3 subunits lead to impaired biogenesis of lysosome-related organelles (LROs) such as mammalian melanosomes and insect eye pigment granules. In this work, we have performed a forward screening for genetic modifiers of AP-3 function in the fruit fly, Drosophila melanogaster. Specifically, we have tested collections of large multi-gene deletions--which together covered most of the autosomal chromosomes-to identify chromosomal regions that, when deleted in single copy, enhanced or ameliorated the eye pigmentation phenotype of two independent AP-3 subunit mutants. Fine-mapping led us to define two non-overlapping, relatively small critical regions within fly chromosome 3. The first critical region included the Atg2 gene, which encodes a conserved protein involved in autophagy. Loss of one functional copy of Atg2 ameliorated the pigmentation defects of mutants in AP-3 subunits as well as in two other genes previously implicated in LRO biogenesis, namely Blos1 and lightoid, and even increased the eye pigment content of wild-type flies. The second critical region included the ArfGAP1 gene, which encodes a conserved GTPase-activating protein with specificity towards GTPases of the Arf family. Loss of a single functional copy of the ArfGAP1 gene ameliorated the pigmentation phenotype of AP-3 mutants but did not to modify the eye pigmentation of wild-type flies or mutants in Blos1 or lightoid. Strikingly, loss of the second functional copy of the gene did not modify the phenotype of AP-3 mutants any further but elicited early lethality in males and abnormal eye morphology when combined with mutations in Blos1 and lightoid, respectively. These results provide genetic evidence for new functional links connecting the machinery for biogenesis of LROs with molecules implicated in autophagy and small GTPase regulation.

Show MeSH
Related in: MedlinePlus