Limits...
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.

Show MeSH

Related in: MedlinePlus

Attempts to validate selected deficiencies carrying the w+mC marker as genetic modifiers of the g2 mutation.(A) Red pigments were extracted from the heads of AP-3-deficient (g2) or White-negative (w1118) adult males carrying single copies of the indicated deficiencies with their associated w+mC marker. 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 2–10 biological replicates. Notice that the activity of the w+mC marker associated with deficiency Df(3L)ED4978 resulted in a red pigment content (arrow) higher than that of g2 flies (black bar). (B-D) Analyses of red pigment content in the eyes of adult g2 mutant males carrying no deletions (—), single copies of the deficiencies Df(3L)ED4978 (B), Df(3R)Exel6195 (C) and Df(2L)XE-3801 (D) that had been identified through screening, or single copies of deficiencies with partially overlapping deletions and devoid of the w+mC marker. Schematic representations of the extent of overlap between the chromosomal regions deleted in the deficiencies identified through screening (blue) and the others (grey) are included in each figure panel. Notice in (C) that a small portion of the chromosomal region deleted in Df(3R)Exel6195 (dashed box) did not overlap with any of those deleted in other available deficiencies. One-way ANOVA followed by Dunnett’s test of each group versus control g2 flies carrying no deletion (black bars): **p<0.01; ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4643998&req=5

pone.0143026.g003: Attempts to validate selected deficiencies carrying the w+mC marker as genetic modifiers of the g2 mutation.(A) Red pigments were extracted from the heads of AP-3-deficient (g2) or White-negative (w1118) adult males carrying single copies of the indicated deficiencies with their associated w+mC marker. 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 2–10 biological replicates. Notice that the activity of the w+mC marker associated with deficiency Df(3L)ED4978 resulted in a red pigment content (arrow) higher than that of g2 flies (black bar). (B-D) Analyses of red pigment content in the eyes of adult g2 mutant males carrying no deletions (—), single copies of the deficiencies Df(3L)ED4978 (B), Df(3R)Exel6195 (C) and Df(2L)XE-3801 (D) that had been identified through screening, or single copies of deficiencies with partially overlapping deletions and devoid of the w+mC marker. Schematic representations of the extent of overlap between the chromosomal regions deleted in the deficiencies identified through screening (blue) and the others (grey) are included in each figure panel. Notice in (C) that a small portion of the chromosomal region deleted in Df(3R)Exel6195 (dashed box) did not overlap with any of those deleted in other available deficiencies. One-way ANOVA followed by Dunnett’s test of each group versus control g2 flies carrying no deletion (black bars): **p<0.01; ***p<0.001.

Mentions: Three of the five deficiencies selected for validation, namely Df(2L)XE-3801, Df(3R)Exel6195, and Df(3L)ED4978, carried the construct mini-white (w+mC) as a genetic marker. It is well known that expression of this construct, which represents a truncated version of the w gene lacking some regulatory sequences, varies significantly depending upon the site of chromosomal insertion [57]. Given that high levels of White protein overexpression had been reported to increase, albeit modestly, eye pigmentation of AP-3 δ mutants [41], the possibility of ‘false-positive’ hits in our screening owing to high expression of the w+mC construct deserved consideration. To begin to address this issue, genetic crosses were set up to generate male flies hemizygous for the White- allele w1118 (on chromosome X) and heterozygous for each of the three deficiencies; in these flies, White function derived exclusively from expression of the w+mC construct. As shown in Fig 3A (arrow), the w+mC construct carried by one of the three deficiencies, Df(3L)ED4978, on a White- background resulted in red pigment levels of almost 40% of wild-type levels. In further support of the notion that deficiency Df(3L)ED4978 might have represented a false-positive hit in our screening, none of two ‘overlapping’ deficiencies–with deletions that together covered the entire region deleted in Df(3L)ED4978 –modified the eye pigmentation phenotype of g2 flies (Fig 3B). Although the w+mC construct carried by the other two deficiencies, Df(2L)XE-3801 and Df(3R)Exel6195, resulted in very little pigmentation on a White- background (Fig 3A), attempts to validate their phenotypic modifier effect observed on the g2 background, using independent overlapping deficiencies, were nonetheless unsuccessful (Fig 3C and 3D). It should be noted, however, that the deficiencies available for validation did not completely cover the genomic region deleted in Df(3R)Exel6195, thus leaving open the possibility that the partial phenotypic suppression effect elicited by this deficiency could have been caused by hemizygous deletion of a gene in the region that was not deleted in any of the other deficiencies tested (Fig 3C, dashed rectangle). Interestingly, this region turned out to contain a single gene, CG31145, which was reported to encode a Golgi-localized protein with casein-kinase activity [58]. Further investigation will be required to test the possibility of CG31145 being a genetic modifier of AP-3 function in the fly eye.


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)

Attempts to validate selected deficiencies carrying the w+mC marker as genetic modifiers of the g2 mutation.(A) Red pigments were extracted from the heads of AP-3-deficient (g2) or White-negative (w1118) adult males carrying single copies of the indicated deficiencies with their associated w+mC marker. 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 2–10 biological replicates. Notice that the activity of the w+mC marker associated with deficiency Df(3L)ED4978 resulted in a red pigment content (arrow) higher than that of g2 flies (black bar). (B-D) Analyses of red pigment content in the eyes of adult g2 mutant males carrying no deletions (—), single copies of the deficiencies Df(3L)ED4978 (B), Df(3R)Exel6195 (C) and Df(2L)XE-3801 (D) that had been identified through screening, or single copies of deficiencies with partially overlapping deletions and devoid of the w+mC marker. Schematic representations of the extent of overlap between the chromosomal regions deleted in the deficiencies identified through screening (blue) and the others (grey) are included in each figure panel. Notice in (C) that a small portion of the chromosomal region deleted in Df(3R)Exel6195 (dashed box) did not overlap with any of those deleted in other available deficiencies. One-way ANOVA followed by Dunnett’s test of each group versus control g2 flies carrying no deletion (black bars): **p<0.01; ***p<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0143026.g003: Attempts to validate selected deficiencies carrying the w+mC marker as genetic modifiers of the g2 mutation.(A) Red pigments were extracted from the heads of AP-3-deficient (g2) or White-negative (w1118) adult males carrying single copies of the indicated deficiencies with their associated w+mC marker. 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 2–10 biological replicates. Notice that the activity of the w+mC marker associated with deficiency Df(3L)ED4978 resulted in a red pigment content (arrow) higher than that of g2 flies (black bar). (B-D) Analyses of red pigment content in the eyes of adult g2 mutant males carrying no deletions (—), single copies of the deficiencies Df(3L)ED4978 (B), Df(3R)Exel6195 (C) and Df(2L)XE-3801 (D) that had been identified through screening, or single copies of deficiencies with partially overlapping deletions and devoid of the w+mC marker. Schematic representations of the extent of overlap between the chromosomal regions deleted in the deficiencies identified through screening (blue) and the others (grey) are included in each figure panel. Notice in (C) that a small portion of the chromosomal region deleted in Df(3R)Exel6195 (dashed box) did not overlap with any of those deleted in other available deficiencies. One-way ANOVA followed by Dunnett’s test of each group versus control g2 flies carrying no deletion (black bars): **p<0.01; ***p<0.001.
Mentions: Three of the five deficiencies selected for validation, namely Df(2L)XE-3801, Df(3R)Exel6195, and Df(3L)ED4978, carried the construct mini-white (w+mC) as a genetic marker. It is well known that expression of this construct, which represents a truncated version of the w gene lacking some regulatory sequences, varies significantly depending upon the site of chromosomal insertion [57]. Given that high levels of White protein overexpression had been reported to increase, albeit modestly, eye pigmentation of AP-3 δ mutants [41], the possibility of ‘false-positive’ hits in our screening owing to high expression of the w+mC construct deserved consideration. To begin to address this issue, genetic crosses were set up to generate male flies hemizygous for the White- allele w1118 (on chromosome X) and heterozygous for each of the three deficiencies; in these flies, White function derived exclusively from expression of the w+mC construct. As shown in Fig 3A (arrow), the w+mC construct carried by one of the three deficiencies, Df(3L)ED4978, on a White- background resulted in red pigment levels of almost 40% of wild-type levels. In further support of the notion that deficiency Df(3L)ED4978 might have represented a false-positive hit in our screening, none of two ‘overlapping’ deficiencies–with deletions that together covered the entire region deleted in Df(3L)ED4978 –modified the eye pigmentation phenotype of g2 flies (Fig 3B). Although the w+mC construct carried by the other two deficiencies, Df(2L)XE-3801 and Df(3R)Exel6195, resulted in very little pigmentation on a White- background (Fig 3A), attempts to validate their phenotypic modifier effect observed on the g2 background, using independent overlapping deficiencies, were nonetheless unsuccessful (Fig 3C and 3D). It should be noted, however, that the deficiencies available for validation did not completely cover the genomic region deleted in Df(3R)Exel6195, thus leaving open the possibility that the partial phenotypic suppression effect elicited by this deficiency could have been caused by hemizygous deletion of a gene in the region that was not deleted in any of the other deficiencies tested (Fig 3C, dashed rectangle). Interestingly, this region turned out to contain a single gene, CG31145, which was reported to encode a Golgi-localized protein with casein-kinase activity [58]. Further investigation will be required to test the possibility of CG31145 being a genetic modifier of AP-3 function in the fly eye.

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