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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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ArfGAP1 as a modifier of the AP-3 eye pigmentation phenotype.(A-C) Red pigments were extracted from the heads of adult male flies of the indicated genetic backgrounds carrying wild-type (+) or mutant (G3-85) alleles of the ArfGAP1 gene on chromosome 3. The extracted pigments were quantified as described under Materials and Methods, and the resulting values expressed as percentages of the pigment content of wild-type (Canton-S) flies. Bars represent means + SD of 3–17 biological replicates. Statistical analyses were performed by means of Student’s t-test (A and B) or one-way ANOVA followed by Dunnett’s test of each group versus g2 flies carrying no deletion (C): *p<0.05, **p<0.01, ***p<0.001. Notice that a single copy of ArfGAP1G3-85 mutant allele over wild-type ArfGAP1 was sufficient to ameliorate the pigmentation defects of both g2 (A) and rb1 (B) AP-3-subunit mutants. Notice in (C) that such partial suppressor effect was not exacerbated in flies homozygous for the ArfGAP1G3-85 allele or heterozygous for this allele over any of two deficiencies in which the deleted genomic regions include the entire ArfGAP1 gene, namely Df(3L)eygC1 (Df1) and Df(3L)BSC380 (Df2). The deficiency Df(3L)BSC413, in which the deleting genomic region excludes the ArfGAP1 gene, was used as a control (Df3).
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pone.0143026.g007: ArfGAP1 as a modifier of the AP-3 eye pigmentation phenotype.(A-C) Red pigments were extracted from the heads of adult male flies of the indicated genetic backgrounds carrying wild-type (+) or mutant (G3-85) alleles of the ArfGAP1 gene on chromosome 3. The extracted pigments were quantified as described under Materials and Methods, and the resulting values expressed as percentages of the pigment content of wild-type (Canton-S) flies. Bars represent means + SD of 3–17 biological replicates. Statistical analyses were performed by means of Student’s t-test (A and B) or one-way ANOVA followed by Dunnett’s test of each group versus g2 flies carrying no deletion (C): *p<0.05, **p<0.01, ***p<0.001. Notice that a single copy of ArfGAP1G3-85 mutant allele over wild-type ArfGAP1 was sufficient to ameliorate the pigmentation defects of both g2 (A) and rb1 (B) AP-3-subunit mutants. Notice in (C) that such partial suppressor effect was not exacerbated in flies homozygous for the ArfGAP1G3-85 allele or heterozygous for this allele over any of two deficiencies in which the deleted genomic regions include the entire ArfGAP1 gene, namely Df(3L)eygC1 (Df1) and Df(3L)BSC380 (Df2). The deficiency Df(3L)BSC413, in which the deleting genomic region excludes the ArfGAP1 gene, was used as a control (Df3).

Mentions: The second candidate was ArfGAP1 (formerly known as Gap69C), which encodes a conserved GTPase-activating protein (GAP) with specificity to promote hydrolysis of GTP bound to members of the Arf protein family [52,86]. Flies carrying the allele ArfGAP1G3-85, either in homozygous or heterozygous form, are viable and display no eye morphological abnormalities [52]. Here, a single copy of the ArfGAP1G3-85 allele elicited statistically significant increases in the pigment contents of the AP-3 mutants g2 (Fig 7A) and rb1 (Fig 7B). On the other hand, and consistently with the behavior observed for the Df(3L)eygC1 deficiency (Table 1), a single copy of the ArfGAP1G3-85 allele failed to modify the red pigment content of wild-type flies (Fig 8A). Interestingly, the modifier effect elicited by the ArfGAP1G3-85 allele in the g2 background appeared to follow a dominant pattern. Thus, eye pigmentation increases similar to those observed for g2 flies heterozygous for ArfGAP1G3-85 over a normal third chromosome (Fig 7A) were observed not only for g2 flies that were heterozygous for ArfGAP1G3-85 over a control deletion (referred in the figure to as ‘Df3’) but also for those that were homozygous for the ArfGAP1G3-85 mutation or compound heterozygous for ArfGAP1G3-85 over either of two large deletions (‘Df1’ and ‘Df2’) encompassing the entire ArfGAP1 gene (Fig 7C).


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)

ArfGAP1 as a modifier of the AP-3 eye pigmentation phenotype.(A-C) Red pigments were extracted from the heads of adult male flies of the indicated genetic backgrounds carrying wild-type (+) or mutant (G3-85) alleles of the ArfGAP1 gene on chromosome 3. The extracted pigments were quantified as described under Materials and Methods, and the resulting values expressed as percentages of the pigment content of wild-type (Canton-S) flies. Bars represent means + SD of 3–17 biological replicates. Statistical analyses were performed by means of Student’s t-test (A and B) or one-way ANOVA followed by Dunnett’s test of each group versus g2 flies carrying no deletion (C): *p<0.05, **p<0.01, ***p<0.001. Notice that a single copy of ArfGAP1G3-85 mutant allele over wild-type ArfGAP1 was sufficient to ameliorate the pigmentation defects of both g2 (A) and rb1 (B) AP-3-subunit mutants. Notice in (C) that such partial suppressor effect was not exacerbated in flies homozygous for the ArfGAP1G3-85 allele or heterozygous for this allele over any of two deficiencies in which the deleted genomic regions include the entire ArfGAP1 gene, namely Df(3L)eygC1 (Df1) and Df(3L)BSC380 (Df2). The deficiency Df(3L)BSC413, in which the deleting genomic region excludes the ArfGAP1 gene, was used as a control (Df3).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4643998&req=5

pone.0143026.g007: ArfGAP1 as a modifier of the AP-3 eye pigmentation phenotype.(A-C) Red pigments were extracted from the heads of adult male flies of the indicated genetic backgrounds carrying wild-type (+) or mutant (G3-85) alleles of the ArfGAP1 gene on chromosome 3. The extracted pigments were quantified as described under Materials and Methods, and the resulting values expressed as percentages of the pigment content of wild-type (Canton-S) flies. Bars represent means + SD of 3–17 biological replicates. Statistical analyses were performed by means of Student’s t-test (A and B) or one-way ANOVA followed by Dunnett’s test of each group versus g2 flies carrying no deletion (C): *p<0.05, **p<0.01, ***p<0.001. Notice that a single copy of ArfGAP1G3-85 mutant allele over wild-type ArfGAP1 was sufficient to ameliorate the pigmentation defects of both g2 (A) and rb1 (B) AP-3-subunit mutants. Notice in (C) that such partial suppressor effect was not exacerbated in flies homozygous for the ArfGAP1G3-85 allele or heterozygous for this allele over any of two deficiencies in which the deleted genomic regions include the entire ArfGAP1 gene, namely Df(3L)eygC1 (Df1) and Df(3L)BSC380 (Df2). The deficiency Df(3L)BSC413, in which the deleting genomic region excludes the ArfGAP1 gene, was used as a control (Df3).
Mentions: The second candidate was ArfGAP1 (formerly known as Gap69C), which encodes a conserved GTPase-activating protein (GAP) with specificity to promote hydrolysis of GTP bound to members of the Arf protein family [52,86]. Flies carrying the allele ArfGAP1G3-85, either in homozygous or heterozygous form, are viable and display no eye morphological abnormalities [52]. Here, a single copy of the ArfGAP1G3-85 allele elicited statistically significant increases in the pigment contents of the AP-3 mutants g2 (Fig 7A) and rb1 (Fig 7B). On the other hand, and consistently with the behavior observed for the Df(3L)eygC1 deficiency (Table 1), a single copy of the ArfGAP1G3-85 allele failed to modify the red pigment content of wild-type flies (Fig 8A). Interestingly, the modifier effect elicited by the ArfGAP1G3-85 allele in the g2 background appeared to follow a dominant pattern. Thus, eye pigmentation increases similar to those observed for g2 flies heterozygous for ArfGAP1G3-85 over a normal third chromosome (Fig 7A) were observed not only for g2 flies that were heterozygous for ArfGAP1G3-85 over a control deletion (referred in the figure to as ‘Df3’) but also for those that were homozygous for the ArfGAP1G3-85 mutation or compound heterozygous for ArfGAP1G3-85 over either of two large deletions (‘Df1’ and ‘Df2’) encompassing the entire ArfGAP1 gene (Fig 7C).

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