Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development

Abstract

Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be substantially reduced by RNAi-mediated silencing of cathepsin L. In contrast to mammalian cells, lysosomal delivery of dCREG does not depend on its carbohydrate moiety. Furthermore, depletion of the putative D. melanogaster lysosomal sorting receptor lysosomal enzyme receptor protein did not compromise cellular retention of dCREG. We also investigated the developmental consequences of dCREG ablation in whole D. melanogaster flies. Ubiquitous depletion of dCREG proved lethal at the late pupal stage once a knock-down efficiency of >95% was achieved. These results demonstrate that dCREG is essential for proper completion of fly development.

Keywords: CREG; Cathepsin; Lysosome; Mannose 6-phosphate; Protein targeting; Proteolytic maturation.

Fig. 1

Posttranslational modification of dCREG by proteolytic maturation and N-glycosylation. (A) D. melanogaster S2 cells overexpressing dCREG under the control of the metallothionein promoter (S2-dCREG) were incubated for 72 h in the presence of 0.5 mM CuSO₄. Cell lysates (C; 6 μg) and media (M) corresponding to 0.15 μg cellular protein were treated for 1 h in the presence (+) or absence (−) of PNGase F. Proteins were then subjected to immunoblotting analysis with antibodies to dCREG. (B) S2 cells expressing V5His-tagged dCREG (S2-dCREG-V5His) and S2 cells expressing V5His-tagged, N-glycosylation-minus dCREG (S2-dCREG-Nmut-V5His) were incubated for 72 h in the presence of 0.5 mM CuSO₄. Cell extracts (C; 6 μg) and conditioned media (M) corresponding to 0.15 μg cellular protein were incubated with (+) or without (−) PNGase F as outlined above, immunoblotted and probed with antibodies to dCREG. The migration positions of selected molecular mass standards are indicated. The results shown are representative of three independent experiments.

Fig. 2

CREG can be proteolytically processed by lysosomal cysteine proteinases. (A) Recombinant mouse CREG (msCREG) and dCREG were incubated with cathepsins B or L for up to 4 h. Samples were then analyzed by SDS-PAGE followed by Coomassie Brilliant Blue staining. (B) Microsomal extracts of S2-dCREG cells (10 μg) were deglycosylated with PNGase F. N-terminally tagged dCREG produced in E. coli (30 ng) was subjected to in vitro processing by cathepsin L for 4 h. The samples were separated by SDS-PAGE and analyzed by immunoblotting with antibodies to dCREG. The migration positions of selected molecular mass standards are indicated.

Fig. 3

Effects of RNAi-mediated knock-down of cathepsin L on dCREG processing and turnover in S2 cells. S2-dCREG cells were induced with 0.5 mM CuSO₄ and incubated with double-stranded (ds) cathepsin L RNA for 72 h. Untreated cells cultivated under the same conditions were used as controls. Cell lysates (6 μg) were deglycosylated with PNGase F and subjected to SDS-PAGE. Immunoblotting was then performed with antibodies to dCREG and cathepsin L. Tubulin served as loading control. Band intensities were determined by densitometry. Data are expressed as means of 3 independent experiments. The migration positions of selected molecular mass standards are indicated. proCREG, full-length dCREG; CREG, processed dCREG; proCL, procathepsin L; mCL, mature cathepsin L. *p ≤ 0.001 (comparison of knock-down cells and controls).

Fig. 4

dCREG co-localizes with lysosomal markers. S2 cells stably transfected with a cDNA construct encoding GFP fused to the C-terminus of dCREG (dCREG-GFP) were stained with Lysotracker Red and analyzed by live-cell imaging (top). S2 cells expressing dCREG-GFP were fixed, permeabilized and incubated with antibodies to cathepsin L (middle) or Golgi-mannosidase II (GMII; bottom). Bound antibodies were then detected with Cy3-labeled secondary antibodies followed by confocal laser-scanning microscopy. Co-localization was assessed by merging of the individual images. Bars, 10 μm.

Fig. 5

LERP is not critical for lysosomal sorting of dCREG. (A) S2-dCREG cells were treated with double-stranded (ds) LERP RNA for 72 h. Untreated cells cultivated under the same conditions were used as controls. Cell lysates (12 μg) were treated with PNGase F prior to immunoblotting with antibodies to LERP. Tubulin served as loading control. Band intensities were determined by densitometry. Data are expressed as normalized means of three independent experiments. (B) Cell lysates (C; 6 μg) and conditioned media (M) corresponding to 0.15 μg (dCREG) or 1.2 μg (cathepsin L) cellular protein were analyzed by immunoblotting with the indicated antibodies after enzymatic deglycosylation where appropriate (dCREG). proCREG, full-length dCREG; CREG, processed dCREG; proCL, procathepsin L; mCL, mature cathepsin L. The migration positions of selected molecular mass standards are indicated. The results shown are representative of three independent experiments.

Fig. 6

Cathepsin L knock-down impairs turnover of dCREG in vivo. Extracts from daughterless-GAL4 (da-GAL4) × w¹¹¹⁸ (control), da-GAL4 > UAS-CB-RNAi^KK and da-GAL4 > UAS-CL-RNAi^KK flies were immunoblotted and probed with antibodies to dCREG (top), cathepsin L (middle) and tubulin as loading control (bottom). Band intensities were determined by densitometry. The cathepsin L and dCREG contents of the respective knock-down flies were calculated relative to control animals. Data are expressed as means of four independent experiments. The migration positions of selected molecular mass standards are indicated. proCREG, full-length dCREG; CREG, processed dCREG; CL, cathepsin L; CB, cathepsin B.

Fig. 7

dCREG depletion in D. melanogaster flies. (A) Double homozygous UAS-CREG-RNAi; da-GAL4 and wild-type (w¹¹¹⁸) flies were propagated at 18 °C or shifted to 25 °C and supplied with fresh media daily for 5 consecutive days. The percentage of eclosed flies was calculated. (B) CREG knock-down efficiency is dependent on temperature and gene dosage. Control (da-GAL4) and UAS-CREG-RNAi/CyO; da-GAL4/TM3, Ser flies were grown at 18 °C or 25 °C. Freshly eclosed flies were subjected to immunoblotting analysis with antibodies to dCREG and tubulin as loading control. Relative dCREG levels were determined by densitometry. Data are expressed as means of three independent experiments.

Fig. 8

Rescue of dCREG knock-down flies by dCREG-GFP expression. (A) Lysates of da-GAL4 (control) and If/CyO; da-GAL4 > UAS-CREG-GFP flies were immunoblotted with antibodies to dCREG and tubulin as loading control. (B) Progeny of UAS-CREG-RNAi/CyO; da-GAL4/TM3, Ser and UAS-CREG-RNAi/CyO; da-GAL4 > UAS-CREG-GFP/TM3, Ser was counted (500–1000 flies per assay). Data from three independent experiments are expressed as means ± SE. Lysates from control, dCREG knock-down (UAS-CREG-RNAi; da-GAL4/TM3, Ser) and rescue flies (UAS-CREG-RNAi; da-GAL4 > UAS-CREG-GFP) flies were subjected to immunoblot analysis with antibodies to dCREG and tubulin as loading control.

Fig. 9

Reduced Lysotracker accumulation in fat bodies of dCREG knock-down flies. Control (da-GAL4 × Canton-S) and da-GAL4 > UAS-CREG-RNAi larvae were either starved for 4 h or further maintained on regular food (fed). Fat bodies were then removed and stained with Lysotracker Red prior to analysis by confocal laser-scanning microscopy. Bars, 20 μm.

Fig. 10

Autophagosome formation is not impaired in dCREG knock-down flies. Control (Lsp2-GAL4-UAS-GFP-Atg8 × Canton-S) and Lsp2-GAL4-UAS-GFP-Atg8 × UAS-CREG-RNAi larvae were either starved for 4 h or further maintained on regular food (fed). Fat bodies were then removed and stained with Lysotracker Red. Acidic compartments (Lysotracker) and autophagosomes (GFP-Atg8) were detected by confocal laser-scanning microscopy. Co-localization was assessed by merging of the individual images. Bars, 20 μm.