The tobacco phosphatidylethanolamine-binding protein NtFT4 increases the lifespan of Drosophila melanogaster by interacting with the proteostasis network

Abstract

Proteostasis reflects the well-balanced synthesis, trafficking and degradation of cellular proteins. This is a fundamental aspect of the dynamic cellular proteome, which integrates multiple signaling pathways, but it becomes increasingly error-prone during aging. Phosphatidylethanolamine-binding proteins (PEBPs) are highly conserved regulators of signaling networks and could therefore affect aging-related processes. To test this hypothesis, we expressed PEPBs in a heterologous context to determine their ectopic activity. We found that heterologous expression of the tobacco (Nicotiana tabacum) PEBP NtFT4 in Drosophila melanogaster significantly increased the lifespan of adult flies and reduced age-related locomotor decline. Similarly, overexpression of the Drosophila ortholog CG7054 increased longevity, whereas its suppression by RNA interference had the opposite effect. In tobacco, NtFT4 acts as a floral regulator by integrating environmental and intrinsic stimuli to promote the transition to reproductive growth. In Drosophila, NtFT4 engaged distinct targets related to proteostasis, such as HSP26. In older flies, it also prolonged Hsp26 gene expression, which promotes longevity by maintaining protein integrity. In NtFT4-transgenic flies, we identified deregulated genes encoding proteases that may contribute to proteome stability at equilibrium. Our results demonstrate that the expression of NtFT4 influences multiple aspects of the proteome maintenance system via both physical interactions and transcriptional regulation, potentially explaining the aging-related phenotypes we observed.

Keywords: aging; chaperone; heat shock proteins; locomotor activity; proteostasis.

Figure 1

Expression of animal PEBPs in tobacco and Arabidopsis. (A) Bimolecular fluorescence complementation (BiFC) in infiltrated Nicotiana benthamiana leaves, representatively showing the interaction between Drosophila PEBP (NmRFP-CG7054) and NtFD1 (CmRFP-NtFD1). (B) BiFC representatively showing the interaction between Drosophila PEBP (NmRFP-CG7054) and tobacco 14-3-3 c (CmRFP-14-3-3 c). Scale bar = 50 μm. (C) Flowering time of tobacco lines expressing PEBP1, CG7054, CG7054-DS, RKIP or hPEBP4 under the control of the cauliflower mosaic virus 35S promoter. Abbreviation: VC: vector control. Flowering time was measured under long-day (LD) conditions in days after potting (dap). Data are means ± SEM, n = 50 (PEBP1, CG7054, CG7054-DS, RKIP and hPEBP4), n = 10 (VC). Significance was tested by one-way ANOVA and Tukey’s post hoc test (b significant compared with PEBP1, c significant compared with CG7054, all other comparisons non-significant). (D) Representative image of a transgenic tobacco plant expressing RKIP compared with the VC. Flowering time (E) and rosette leaf number at the onset of flowering (F) of transgenic Arabidopsis lines expressing RKIP, hPEBP4, CG7054, PEBP1 or the floral inducer NtFT4 under the control of the quadruple cauliflower 35S promoter. Col-0 = wild type A. thaliana Col-0 ecotype used for transformation. Flowering time was measured under LD conditions in days after seeding (das). Data are means ± SEM, n = 30 (CG7054, CG19594), n = 29 (hPEBP4), n = 19 (RKIP), n = 10 (Col-0), n = 8 (NtFT4); ^****p < 0.001 in all pairwise comparisons with NtFT4 (a significant compared with Col-0 (p = 0.091) with all other comparisons being non-significant). Abbreviation: NS: no significant differences in any pairwise comparison. All p-values are provided in Supplementary Table 9. (G) Representative images of transgenic Arabidopsis plants expressing different PEBPs. Col-0 wild type plants (far right), and early flowering Q35-S:NtFT4 (left) and late flowering Q35-S:NtFT2 (far left) plants are shown in comparison with plants expressing the animal PEBPs.

Figure 2

Survival of Drosophila populations expressing NtFT4, NtFT2, CG7054 or CG7054^dsRNA under the control of the daughterless (da) promoter. Survival curves of female (left) and male (right) flies in the filial generation after mating UAS-NtFT4, UAS-NtFT2, UAS-CG7054 or UASt-CG7054^dsRNA with the da-Gal4 driver strain. (A, B) Effect on lifespan of flies constitutively expressing the floral inducer NtFT4 (A) or the floral repressor NtFT2 (B) compared with da-Gal4 x Oregon-R (n = 200). (C, D) Effect on lifespan of flies constitutively expressing the Drosophila PEBP CG7054 (C) or constitutively silencing CG7054 after mating UASt-CG7054^dsRNA with the da-Gal4 driver strain (D) compared with da-Gal4 x Oregon-R (n = 200). Median and mean lifespans and statistical evaluation are summarized in Table 1 (female flies) and Supplementary Table 1 (male flies).

Figure 3

Locomotor behavior of long-lived (da > NtFT4) or short-lived (da > CG7054^dsRNA) Drosophila populations at different ages. Rapid iterative negative geotaxis (RING) assay with virgin female (A) and male (B) flies at 10, 30 or 45 days old. The locomotor behavior was analyzed in the filial generation after mating Oregon-R (control, black), UAS-NtFT4 (green) or UASt-CG7054^dsRNA (blue) flies with the da-Gal4 driver strain. Negative geotaxis was plotted as average velocity (mm/10 s) and was traced for all tracks traveled in the population (numbers below plots). Significance was tested by one-way ANOVA and Tukey’s post hoc test between control, da > NtFT4 and da > CG7054^dsRNA (^****p < 0.001, Abbreviation: NS: not significant). All p-values are provided in Supplementary Table 9.

Figure 4

Interaction partners of NtFT4 identified in immunoprecipitated protein complexes after transient expression in S2 cells. The abundance of the interaction partners was confirmed by immunodetection using mouse anti-Myc (top) or rabbit anti-HA (bottom) antibodies in the extracts and successful precipitation with magnetic anti HA-beads was confirmed by the detection of HA-EGFP-NtFT4 in the eluates. (A) Western blots of extracts (Input) and eluates after co-immunoprecipitation (Eluate) following transient co-transfection of S2 cells with HA-EGFP-NtFT4 plus Myc-Tsn, Myc-14-3-3 ζ, Myc-CG4364, Myc-Df31, Myc-Rack1, Myc-CCT7, Myc-PyK, Myc-CCT2, Myc-Hsp26, Myc-Pen, Myc-Cbs or Myc-p47. Detection of co-immunoprecipitated proteins in the eluate is indicated by red arrowheads. Df31 was not detected in extracts under the mild conditions used for immunoprecipitation (empty arrowhead). (B) Analysis of FRET efficiency in co-transfected cells expressing the donors Cerulean (Cer, negative control), Cer-NtFT4 or Cer-CG7054 plus the acceptors EYFP-CCT7, EYFP-CG4364, EYFP-Df31, EYFP-Hsp26, EXFP-p47, EYFP-Pen, EYFP-PyK or EYFP-Tsn by flow cytometry. Gating strategy and representative controls are shown in Supplementary Figure 8. Cer-NtFT4 and Cer-CG7054 were co-transfected in three independent triplicates (n = 3) and statistical significance was tested by one-sample t-test (^****p < 0.001, ^***p < 0.01, ^**p < 0.05, ^*p < 0.1, Abbreviation: NS: not significant).

Figure 5

Transfection and heat stress response of heat shock proteins in S2 cells. Expression of stress-responsive (Hsp22, Hsp23, Hsp26, Hsp27, Hsp70Aa and Hsc70–4) (A) and non-responsive (HspB8 and l(2)efl) (B) heat shock protein genes in S2 cells after transient transfection with HA-EGFP, HA-NtFT4 or HA-EGFP-NtFT4 compared to non-transfected cells. After transfection and induction of gene expression, cells were cultivated at 27°C (–, white bars) or stressed by heat shock at 37°C for 1 h (+, gray bars show controls and red bars show NtFT4). Relative gene expression was analyzed by qRT-PCR using Gapdh2 as a reference. Data are means ± SEM (n = 3). Significance was tested by one-way ANOVA and Tukey’s post hoc test for responses to transfection (untransfected vs. HA-EGFP vs. HA-NtFT4 vs. HA-EGFP-NtFT4; a = significant compared to nontransfected cells, p < 0.1; b = significant compared to nontransfected cells, p < 0.05; Abbreviation: NS: not significant including all remaining comparisons) and using a t-test for pairwise comparisons of individual responses to heat shock (^****p < 0.001, ^***p < 0.01, ^**p < 0.05, ^*p < 0.1, Abbreviation: NS: not significant). (C) Immunodetection of HSP26 following the transient transfection of S2 cells with HA-EGFP, HA-NtFT4 or HA-EGFP-NtFT4 compared with nontransfected cells. The response of HSP26 to transfection and to heat shock at 37°C was analyzed 1 h after treatment by extracting proteins for immunodetection using anti-HSP26 antibodies (top right). The transient expression of HA-EGFP, HA-NtFT4 or HA-EGFP-NtFT4 was confirmed using anti-HA antibodies (bottom, arrowheads). All p-values are provided in Supplementary Table 9.

Figure 6

Expression of heat shock genes during aging in flies expressing NtFT4. Relative expression of two small heat shock protein genes directly associated with aging (Hsp26 and Hsp27) (A), of the two small heat shock protein genes Hsp22 and Hsp23 (B), of larger heat shock protein genes Hsp83, HspB8, Hsc70-3 and Hsc70-4 (C), and of weakly-expressed heat shock protein genes DnaJ-1, l(2)efl, Hsp68 and Hsp70Aa (D) in female da > NtFT4 flies aged 10, 20 and 50 d, compared with da-Gal4 flies by quantitative RT-PCR. Relative expression was calculated using Gapdh2 as a reference gene. Data are means ± SEM (n = 3). Significance was tested by one-way ANOVA and Tukey’s post hoc test for changes during age (10 d vs. 20 d vs. 50 d) and using a t-test for pairwise comparisons between da-Gal4 and da > NtFT4 flies (^***p < 0.01, ^**p < 0.05, ^*p < 0.1, Abbreviation: NS: not significant; a = significant between 10 d and 20 d, b = significant between 10 d and 50 d, c = significant compared between 20 d and 50 d). (E) Western blot showing the detection of HSP26 in protein extracts from female da > NtFT4 flies aged 10, 20 and 50 d, compared with da-Gal4 flies. A representative Western blot is shown for anti-HSP26 and comparable protein loading was ensured by staining with Coomassie Brilliant Blue. (F) Quantification of relative band intensities from three independent Western blot samples from 20 d (highest levels of HSP26 protein) and 50 d old flies. The relative band intensity was measured with imageJ and calculated by referring to the weakest band on each blot (50 d old da-Gal4 flies). Data are means ± SEM (n = 3), p = 0.029 (t-test), Abbreviation: NS: not significant. The p-values of all comparisons are provided in Supplementary Table 9.

Figure 7

GeneChip 2.0 array and gene expression analysis of female flies expressing NtFT4. (A) Protein classes encoded by differentially expressed genes which were identified in the GeneChip Drosophila Genome 2.0 arrays (Affymetrix). We identified 149 genes that were significantly deregulated in female flies expressing NtFT4, 97 of which were mapped in the Panther database, and 63 genes were classified as representing 12 different protein classes. The largest protein classes were PC00262 (metabolite interconversion enzymes, 27 genes) and PC 00260 (protein modifying enzymes, 9 genes). Significance was determined using the paired t-test. Deregulated genes were included with a log₂ fold change > 1.5 and a p-value < 0.05, n = 3. (B–D) Gene expression analysis. Deregulated genes associated with proteolysis (CG1304, Ser6, CG31205, CG31681, CG32277, CG32523, Jon66Ci and Spn47C) (B), annotated as metabolic enzymes (C), or genes which cannot be classified into groups and genes of unknown function (non-classified/unknown function) (D) identified by transcriptome analysis were analyzed individually in 1d (left, blue), 5 d (middle, green) or 10 d (right, red) old female flies expressing NtFT4 (da > NtFT4) compared with control (da-Gal4) flies (black). Relative expression levels were calculated in relation to the reference genes Gapdh2, 14-3-3 ε and RpL32. Data are means ± SEM (n = 3), p-values are based on a t-test of pairwise comparisons between da > NtFT4 and da-Gal4 flies, ^****p < 0.001, ^***p < 0.01, ^**p < 0.05, ^*p < 0.1, Abbreviation: NS: not significant. The p-values of all comparisons are provided in Supplementary Table 9.