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. 2009 Jun 29;185(7):1181-94.
doi: 10.1083/jcb.200904161. Epub 2009 Jun 22.

The Drosophila deoxyhypusine hydroxylase homologue nero and its target eIF5A are required for cell growth and the regulation of autophagy

Prajal H Patel  1 , Mauro Costa-Mattioli, Karen L Schulze, Hugo J Bellen
Affiliations

The Drosophila deoxyhypusine hydroxylase homologue nero and its target eIF5A are required for cell growth and the regulation of autophagy

Prajal H Patel et al. J Cell Biol. .

Abstract

Hypusination is a unique posttranslational modification by which lysine is transformed into the atypical amino acid hypusine. eIF5A (eukaryotic initiation factor 5A) is the only known protein to contain hypusine. In this study, we describe the identification and characterization of nero, the Drosophila melanogaster deoxyhypusine hydroxylase (DOHH) homologue. nero mutations affect cell and organ size, bromodeoxyuridine incorporation, and autophagy. Knockdown of the hypusination target eIF5A via RNA interference causes phenotypes similar to nero mutations. However, loss of nero appears to cause milder phenotypes than loss of eIF5A. This is partially explained through a potential compensatory mechanism by which nero mutant cells up-regulate eIF5A levels. The failure of eIF5A up-regulation to rescue nero mutant phenotypes suggests that hypusination is required for eIF5A function. Furthermore, expression of enzymatically impaired forms of DOHH fails to rescue nero clones, indicating that hypusination activity is important for nero function. Our data also indicate that nero and eIF5A are required for cell growth and affect autophagy and protein synthesis.

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Figures

Figure 1.
Figure 1.
nero mutants disrupt CG2245 and affect bristle size and viability. (A) nero genomic locus. P[lArB]K48 and P[lacW]s1921 are both inserted within CG1910 and in proximity to CG2245 (shown in red). nero1 and nero2 alleles are derived from the excision of P[lacW]s1921 and disrupt the translational start of CG2245 (disrupted black lines). The 1.5-kb genomic rescue construct (shown in blue) rescues nero mutants to viability. (B) nero mutant clones (outlined in black) display a disruption in both the patterning and the size of bristles (genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80). (C) The 1.5-kb genomic rescue construct rescues bristle defects in nero mutant clones (genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; P[CaSper4–1.5 KB nero rescue]/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80). (D) Overexpression of the nero cDNA (CG2245) in nero mutant clones rescues bristle size defects (genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; UAS-nero/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80). (C and D) Rescued mutant bristles are marked by the recessive yellow mutation and appear light brown. (E) The lethal phase of nero mutants as homozygotes, in heteroallelic combinations, or in combination with Df(3R)04661 is L2 lethal. (F) nero encodes a small 302-aa protein composed of two dyads consisting of four HEAT motifs each.
Figure 2.
Figure 2.
Nero localizes to the ER. (A–C) Antibodies generated against Nero specifically detect the Nero protein on both immunohistochemical preparations and Western blots. (A and B) Nero antibody fails to recognize the protein in mutant clones marked by the absence of GFP (genotype: y w hs-FLP; FRT82B nero1/FRT82B Ubi-GFP). White lines mark the clonal boundary. WT, wild type. (C) The Nero antibody detects a single ∼39-kD band in Canton-S (CS) L2 larval protein extracts (first and second lanes) but fails to detect the protein in protein extracts from nerok48-123 (third lane), P[lacW]s1921 (fourth lane), nero1 (fifth lane), and nero2 (sixth lane) L2 larvae on Western blots. Protein lysates from 10 larvae were loaded in each lane except the second lane, in which only five larvae were loaded. Actin was probed as a loading control. Protein standards run along with larval lysates are marked with black bars. Their sizes are recorded in kilodaltons on the left. (D–F) Double labeling of Canton-S third instar wing discs using the Nero antibody and the KDEL antibody shows extensive colocalization, demonstrating that Nero is ER associated. (G–I) Double labeling of Canton-S third instar garland cells using Nero and KDEL antibodies.
Figure 3.
Figure 3.
nero is required for cell growth. (A) The shaft and socket cells that comprise the external sensory organ on the thorax of the adult fly are typically shorter and smaller in nero mutant clones (genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-Gal80). Mutant bristles are marked by the recessive yellow mutation and appear light brown. (B and C) Socket cells observed 24 h APF labeled with Su(H) are smaller than wild-type (WT) socket cells (genotype: y w hs-FLP/+; UAS-FLP/+; FRT82B nero1/C684-GAL4 FRT82B Ubi-GFP M[3]). The clone is marked by the absence of GFP. (D) Epidermal cells on the adult thorax in the vicinity of nero mutant bristles are often smaller in size, as indicated by smaller trichome size and closer trichome spacing (genotype: y w hs-FLP/+; FRT82B nero1/FRT82B Ubi-GFP). (E and F) nero mutant epidermal cells in the thorax 47 h APF are smaller than adjacent wild-type cells. Thoraxes are labeled with Dlg to reveal cell outlines (genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80). The mutant clone is marked positively with GFP. (B, C, E, and F) White lines mark the clonal boundary.
Figure 4.
Figure 4.
nero regulates organ size, cell number, and proliferation. (A and B) nero clones generated in a wild-type background are poorly competitive. Clones are marked with the absence of white+ and thus appear white. (A) Wild-type clones are easily recovered (genotype: y w eyeless-FLP GMR-lacZ/+; FRT82B+/FRT82B w+). (B) nero clones are usually irrecoverable (genotype: y w eyeless-FLP GMR-lacZ/+; FRT82B nero1/FRT82B w+). The white arrow points to a small nero mutant clone at the edge of the eye. (C and D) To provide nero mutant cells a competitive advantage, clones were made using an FRT-bearing chromosome carrying a recessive cell lethal that effectively eliminates wild-type twin spots. Clones are marked as in A and B. (C) Wild-type clones proliferate and take over most of the eye (genotype: y w eyeless-FLP GMR-lacZ/+; FRT82B+/FRT82B w+ l[3]cl). (D) nero clones generated in a cell lethal background can, like wild type, dominate the entire eye (genotype: y w eyeless-FLP GMR-lacZ/+; FRT82B nero1/FRT82B w+ l[3]cl). nero mutant eyes and heads are smaller than wild-type control heads, suggesting a defect in organ size regulation. Eyes are also rough. (E) Cell number in wild-type (WT) clones is roughly similar to cell number in their twin spots (genotype: y w hs-FLP; FRT82B +/FRT82B Ubi-GFP). Black bars correspond to the clone, and gray bars correspond to the twin spot. (F) nero mutant clones have fewer cells than wild type (genotype: y w hs-FLP; FRT82B nero1/FRT82B Ubi-GFP). Black and gray bars are the same as in E. (G and H) nero mutant clones marked positively with GFP incorporate BrdU more poorly than adjacent wild-type cells (genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80). White lines mark the clonal boundary.
Figure 5.
Figure 5.
nero mutants display autophagic starvation response. (A) Fed second instar Canton-S (CS) larvae lack enlarged, acidic autophagic structures, as marked by LysoTracker fluorescence. (B) Second instar Canton-S animals subjected to a 4-h starvation period show LysoTracker-fluorescent autophagic structures. (C) Fed nero1 larvae display autophagic structures despite nutrient availability. (D–F) LysoTracker-fluorescent structures and ATG8b-GFP fusion proteins colocalize in nero1 mutant larvae, demonstrating that these structures are autophagosomes (genotype: hs-ATG8b/+; FRT82B nero1/FRT82B nero1).
Figure 6.
Figure 6.
Loss of eIF5A causes phenotypes reminiscent of nero. (A) Wing imaginal disc in which eIF5A dsRNA has been expressed under the regulation of the dpp-GAL4 driver line and labeled with anti-eIF5A antibody. Anti-eIF5A antibody fails to detect the eIF5A protein in cells expressing eIF5A dsRNA. (B) Wing imaginal disc in which eIF5A under UAS regulation has been overexpressed using dpp-GAL4 and stained with anti-eIF5A antibody. Anti-eIF5A antibody detects the overexpressed eIF5A protein. (C) Anti-eIF5A antibody detects an ∼17-kD band in EGFP dsRNA–treated S2 cells but fails to detect this band in eIF5A dsRNA–treated S2 cells. Actin is used as the loading control. Protein standards run along with S2 cell lysates are marked with black bars. Their sizes are recorded in kilodaltons on the left. (D) Control adult wing (genotype: dpp-GAL/+). (E) Adult wing in which eIF5A dsRNA has been expressed under the regulation of the dpp-Gal4 driver. The distance between the L3 and L4 vein (double-headed arrow) is drastically reduced (genotype: dpp-Gal4/UAS-eIF5A RNAi). (F) Adult wing in which wild-type eIF5A under UAS regulation was overexpressed using dpp-Gal4. The distance between the L3 and L4 vein (double-headed arrow) appears similar to the experimental control (D; genotype: dpp-GAL4/UAS-eIF5A RNAi). (G) RNAi knockdown of eIF5A using eq-GAL4 causes defects in bristle growth similar to nero mutations (genotype: eq-GAL4/+; UAS-eIF5A RNAi). (H) Synchronized larvae 72 h after egg hatching of four different genotypes (from left to right): y w, eIF5AP01296, tub-GAL4/UAS-eIF5A RNAi, and nero1. (I) eIF5AP01296 larval fat bodies undergo constitutive autophagy under fed conditions. (J) RNAi knockdown of eIF5A in the fat body induces constitutive autophagy under fed conditions (genotype: Adh-GAL4/+; UAS-eIF5A RNAi/+). (K) Larval fat bodies of P element revertant eIF5APrev do not undergo autophagy under fed conditions.
Figure 7.
Figure 7.
nero regulates eIF5A levels in vivo and Nero's DOHH activity is required for nero function. (A) Synchronized larvae 72 h after egg hatching of four different genotypes (from left to right): y w, nero1, eIF5AP01296, and eIF5AP01296; nero1. The growth of eIF5AP01296; nero1 double mutant larvae appears to be more impaired than either eIF5AP01296 or nero1 homozygous mutant larvae. (B) Overexpression of eIF5A in nero mutant clones fails to rescue bristle growth defects associated with nero mutant clones (genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; UAS-eIF5A/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80). (C and D) eIF5A levels are dramatically up-regulated in nero mutant clones in the wing imaginal disc. nero mutant clones are marked by the absence of GFP (green; genotype: y w hs-FLP; FRT82B nero1/FRT82B Ubi-GFP). White lines mark the clonal boundary. WT, wild type. (E) Overexpression of hDOHH in nero mutant clones rescues bristle size defects (genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; UAS-hDOHH/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80). (F–I) Overexpression of mutated forms of hDOHH in nero mutant clones fails to rescue bristle size defects. Genotypes are essentially identical to E except mutated forms of hDOHH are expressed under the UAS regulation. (F) Genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; UAS-DOHH H56A/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80. (G) Genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; UAS-hDOHH H89A/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80. (H) Genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; UAS-hDOHH H208A/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80. (I) Genotype: y w hs-FLP tub-GAL4 UAS-GFP-6xMYC-NLS/+; UAS-hDOHH H240A/+; FRT82B nero1/FRT82B hsp70-CD2 y+ tub-GAL80. (B and E–I) Mutant bristles are marked by the recessive yellow mutation and appear light brown.
Figure 8.
Figure 8.
RNA knockdown of either Nero or eIF5A blocks translation elongation in Drosophila S2 cells. Polysome analysis of EGFP, nero, and eIF5A dsRNA–treated S2 cells. Cells were harvested, lysed, and fractionated by centrifugation on a 10–50% sucrose gradient. Polysomes were analyzed as described in Materials and methods. EGFP (left), nero (middle), and eIF5A dsRNA–treated (right) images are shown. The positions of the polysomes and ribosomes are indicated.
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References

    1. Abbruzzese A., Park M.H., Folk J.E. 1986. Deoxyhypusine hydroxylase from rat testis. Partial purification and characterization.J. Biol. Chem. 261:3085–3089 - PubMed
    1. Audibert A., Simon F., Gho M. 2005. Cell cycle diversity involves differential regulation of Cyclin E activity in the Drosophila bristle cell lineage.Development. 132:2287–2297 - PubMed
    1. Brand A.H., Perrimon N. 1993. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.Development. 118:401–415 - PubMed
    1. Byers T.L., Lakanen J.R., Coward J.K., Pegg A.E. 1994. The role of hypusine depletion in cytostasis induced by S-adenosyl-L-methionine decarboxylase inhibition: new evidence provided by 1-methylspermidine and 1,12-dimethylspermine.Biochem. J. 303:363–368 - PMC - PubMed
    1. Chatterjee I., Gross S.R., Kinzy T.G., Chen K.Y. 2006. Rapid depletion of mutant eukaryotic initiation factor 5A at restrictive temperature reveals connections to actin cytoskeleton and cell cycle progression.Mol. Genet. Genomics. 275:264–276 - PubMed

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