Cup is a nucleocytoplasmic shuttling protein that interacts with the eukaryotic translation initiation factor 4E to modulate Drosophila ovary development

Abstract

In Drosophila, the product of the fs (2)cup gene (Cup) is known to be crucial for diverse aspects of female germ-line development. Its functions at the molecular level, however, have remained mainly unexplored. Cup was found to directly associate with eukaryotic translation initiation factor 4E (eIF4E). In this report, we show that Cup is a nucleocytoplasmic shuttling protein and that the interaction with eIF4E promotes retention of the Cup protein in the cytoplasm. Cup is required for the correct accumulation and localization of eIF4E within the posterior cytoplasm of developing oocytes. We furthermore show that cup and eIF4E interact genetically, because a reduction in the level of eIF4E activity deteriorates the development and growth of ovaries bearing homozygous cup mutant alleles. Our results reveal a crucial role for the Cup-eIF4E complex in ovary-specific developmental programs.

Fig. 1.

Cup interacts with eIF4E in yeast and in vitro. (A) eIF4E clones isolated from the yeast two-hybrid screen. Interaction of the various isolated eIF4E fragments (amino acids are indicated on the left) with full-length Cup was determined by streaking transformants on selective media containing 10 mM 3-aminotriazole. (B) eIF4E binds to ovary-derived Cup. GST-eIF4E and GST proteins were challenged with wild-type fly ovary extracts; retained proteins were eluted and separated by SDS/PAGE. An arrow indicates the Cup protein (≈145 kDa).

Fig. 2.

Cup is a nucleocytoplasmic shuttling protein. The upper two rows show that Cup and eIF4E colocalize to the cytoplasm of expressing Drosophila S2 cells. The lower two rows show that nuclear relocalization of Cup is blocked by eIF4E. Transfected S2 cells, with or without LMB treatment, were stained by immunofluorescence with α-Cup and α-HA (detecting HA-tagged eIF4E) antibodies and analyzed by confocal microscopy. Colocalization of Cup and eIF4E appears in yellow. Nuclei were stained with Hoechst.

Fig. 3.

Cup and eIF4E colocalize to the posterior cytoplasm in growing oocytes. (A, B, and E–J) Confocal microscopy of cup²¹/+;eIF4E⁰⁷²³⁸/+ (A, B, E, G, and I) and cup²¹/cup²¹ (F, H, and J) egg chambers to visualize eIF4E (green), Cup (red), and colocalization (yellow). Both cup²¹/cup²¹ and cup²¹/cup²¹;eIF4E⁰⁷²³⁸/+ ovaries show comparable expression of the two proteins. (A and B) Germarium and early egg chambers, respectively. Protein enrichment can be seen mainly in the developing oocyte. (E and F) Stage 3 egg chambers. (G and H) Stage 5 egg chambers. (I and J) Stage 7 egg chambers. Arrowheads indicate the oocytes. Similar images were obtained with single antibodies. (C and D) Stage 8 cup²¹/+;eIF4E⁰⁷²³⁸/+ and cup²¹/cup²¹ egg chambers, respectively, immunostained with α-eIF4E and α-Orb to mark the developing oocyte.

Fig. 4.

Cup interacts with eIF4E (4E) through an eIF4E-binding motif. (A) Cup deletions tested in a yeast two-hybrid assay. Growth on selective media containing 4 mM 3-aminotriazole (+) is indicated on the right. Amino acids 342–453 (Cup100) represent the minimal region of Cup interacting with eIF4E (black box). The striped box indicates the Nos-binding domain (amino acids 593–962) (4). (B) Cup100 interacts with eIF4E in a GST pull-down assay. Retained proteins were eluted and subjected to SDS/PAGE. (C) Coimmunoprecipitation assay using an α-HA antibody of ³⁵S-Met-labeled Cup100 and HA-tagged eIF4E produced in reticulocyte lysates. Cup100 coimmunoprecipitates with HA-eIF4E (Cup100 + 4E). (Right) Control immunoprecipitations with α-HA antibody show specific immunoprecipitation of HA-eIF4E but not of Cup100. (D) Yeast two-hybrid assay using Cup100 (wild-type) and Cup100-MEBS (mutant). Yeast transformants were streaked on selective media containing 4 mM 3-aminotriazole. AD, GAL4 AD; DBD, GAL4 DBD.

Fig. 5.

Cup modulates eIF4E functions. (A) Cup competes with eIF4G (4G-200) for binding to eIF4E (4E). Coimmunoprecipitation assays using an α-HA antibody on HA-tagged eIF4E, Cup100, and a portion of eIF4G produced in reticulocyte extracts. A 2.5× molar excess of eIF4G added to Cup100 and eIF4E reduced the amount of bound Cup100 (lane 5) by 25%; reciprocally, a 1×,3×, and 13× molar excess of Cup100 added to eIF4E and eIF4G-200 reduced the amount of bound eIF4G-200 (lanes 10–12) by a maximum of 45%. Lanes 6–9, control immunoprecipitations. Densitometry was performed by using image-quant software. (B) Effect of phosphatase treatment on the mobility of eIF4E isoforms. Wild-type (lanes 1–4) and cup²¹/cup²¹ (lanes 5–8) ovary extracts were treated with calf intestinal phosphatase (CIP) in the absence (lanes 2 and 6) or presence (lanes 4 and 8) of phosphatase inhibitors (PI). Samples were analyzed by Western blot with α-eIF4E antibody. α-β-Tubulin antibody was used as loading control.

Fig. 6.

cup and eIF4E interact genetically to modulate ovary growth and development. (Upper) Nomarski photomicrographs (all at the same magnification) of ovaries dissected 3–5 days after eclosion. (Lower) Fluorescence photomicrographs (all at the same magnification) of Hoechst-stained ovarioles to visualize DNA. Note that flies carrying heterozygous cup²¹ or eIF4E⁰⁷²³⁸ mutant alleles, as well as a transheterozygous combination of these two alleles, display normal ovaries.