Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation

Abstract

To better understand the response to mitochondrial dysfunction, we examined the mechanism by which ATFS-1 (activating transcription factor associated with stress-1) senses mitochondrial stress and communicates with the nucleus during the mitochondrial unfolded protein response (UPR(mt)) in Caenorhabditis elegans. We found that the key point of regulation is the mitochondrial import efficiency of ATFS-1. In addition to a nuclear localization sequence, ATFS-1 has an N-terminal mitochondrial targeting sequence that is essential for UPR(mt) repression. Normally, ATFS-1 is imported into mitochondria and degraded. However, during mitochondrial stress, we found that import efficiency was reduced, allowing a percentage of ATFS-1 to accumulate in the cytosol and traffic to the nucleus. Our results show that cells monitor mitochondrial import efficiency via ATFS-1 to coordinate the level of mitochondrial dysfunction with the protective transcriptional response.

Figure 1. In the absence of stress, ATFS-1 is imported into mitochondria and degraded

A. ATFS-1 schematic. B. Photomicrographs of HeLa cells expressing ATFS-1^1-100::GFP or GFP stained with MitoTracker. Scale bar, 0.25 mm. C. Immunoblots of HeLa cells expressing GFP or ATFS-1^1-100::GFP following fractionation into total lysate (T), postmitochondrial supernatant (S) and mitochondrial pellet (M). Longer exposure of the ATFS-1^1-100::GFP panel was required due to toxicity and weak expression. D. Immunoblots of atfs-1_pr::atfs-1::gfp, wild-type or atfs-1(tm4525) worms raised on control or lon(RNAi). ATFS-1 (➤) and ATFS-1::GFP (*) are marked. E. Immunoblots of wild-type worms fed control or lon(RNAi) following cellular fractionation. Endogenous NDUFS3 serves as a mitochondrial marker and α-tubulin as a cytosolic marker. F. Photomicrographs of hsp-60_pr::gfp transgenic worms raised on control, lon or spg-7(RNAi). Scale bar, 0.5 mm.

Figure 2. In the presence of mitochondrial stress, unprocessed ATFS-1 accumulates in nuclei

A. Photomicrographs of two intestinal cells in atfs-1_pr::atfs-1::gfp or hsp-16_pr::atfs-1^Δ1-32.myc::gfp transgenic animals raised on control, spg-7 or tim-23(RNAi) or 100 μg/ml EtBr with the nuclei outlined (right panels). The punctae (arrowhead) are endogenous autofluorescence from intestinal cell lysosomes. The mean percentage ± SEM of worms with nuclear accumulation of ATFS-1::GFP is indicated (N = 3). Scale bar, 15 μm. B. Immunoblots of fractionated lysates from wild-type worms raised on control, spg-7(RNAi) or EtBr (100 μg/ml). Lanes 1–9 are 100 μg from the described fractions and lane 10 (*) is 3 μg from the mitochondrial pellet of worms raised on lon(RNAi) for size comparison. Unprocessed and lon(RNAi) stabilized (Ls) ATFS-1 are indicated as are non-specific bands (.). C. Immunoblots of fractionated extracts from wild-type or haf-1(ok705) worms raised on control(RNAi) in the absence or presence of 30 μg/ml EtBr expressing hsp-16_pr::gfp^mt. D. Immunoblots of fractionated extracts from wild-type or haf-1(ok705) worms raised on lon(RNAi) in the absence or presence of 30 μg/ml EtBr expressing hsp-16_pr::atfs-1^FL.

Figure 3. HAF-1 modulates UPR^mt signaling by slowing mitochondrial import of ATFS-1

A. Photomicrographs of wild-type and haf-1(ok705); hsp-60_pr::gfp worms raised on control, tim-23, cco-1(RNAi), EtBr or 0.5 mM paraquat. Scale bar, 0.5 mm. The images for cco-1(RNAi) and paraquat were exposed longer because of smaller worm size. B. Immunoblots of wild-type, clk-1(qm30) or clk-1(qm30); haf-1(ok705) worms raised on control or lon(RNAi). C. Photomicrographs of atfs-1(tm4525); hsp-60_pr::gfp worms expressing wild-type (^FL) ATFS-1, ATFS-1^Δ1-32.myc or ATFS-1^{Δ1-32.myc.ΔNLS} raised on control(RNAi). The lower panel harbors the haf-1(ok705) allele. Scale bar, 0.5 mm. D. Schematic illustrating ATFS-1 regulation.

Figure 4. ATFS-1 mediates a broad and protective transcriptional program

A. Representative photomicrographs of wild-type or isp-1(qm150) worms raised on control or atfs-1(RNAi). Scale bar, 1mm. B. Heat map comparing gene expression patterns of wild-type or atfs-1(tm4525) worms raised on control or spg-7(RNAi). C. Functional categories of the 391 ATFS-1-dependent genes identified by hierarchical clustering. D–G. Expression levels of dnj-10, skn-1, gpd-2, and tim-23 mRNA in wild-type or atfs-1(tm4525) worms raised on control or spg-7(RNAi) determined by qRT-PCR (N = 3, ± SD, p* (student t-test) < 0.05).