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 Activating Transcription Factor associated with Stress-1 (ATFS-1) senses mitochondrial stress and communicates with the nucleus during the mitochondrial unfolded protein response (UPR^mt). We found that the key point of regulation was the mitochondrial import efficiency of ATFS-1. In addition to a nuclear localization sequence, ATFS-1 has an amino-terminal mitochondrial targeting sequence, which was essential for UPR^mt repression. Normally, ATFS-1 is imported into mitochondria and degraded. However, during mitochondrial stress, 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.

Mitochondria import ~99% of their proteome through the TOM (Translocase of the Outer Membrane) and TIM (Translocase of the Inner Membrane) complexes (1, 2). The mitochondrial protein-folding environment is maintained by mitochondrial molecular chaperones whose expression levels are coupled to the state of mitochondrial protein homeostasis by a mitochondria-to-nuclear signaling pathway termed the UPR^mt (3, 4). Evidence in Caenorhabditis elegans implicates the mitochondrial inner membrane peptide transporter, HAF-1, and the bZip transcription factor ATFS-1 in UPR^mt signaling (5).

During mitochondrial stress, ATFS-1 accumulates in the nucleus, because of a nuclear localization signal (NLS). A protein sequence prediction algorithm, Mitoprot II, predicted the presence of an amino-terminal mitochondrial targeting sequence (MTS) as well (6) (Fig. 1A). Indeed, amino acids 1-100 of ATFS-1 were sufficient to target green fluorescent protein (GFP) to HeLa cell mitochondria (Figs. 1B & S1). Consistent with cleavage of the MTS, the mitochondrial enriched form of ATFS-1^1-100::GFP was smaller than the unprocessed form found in the postmitochondrial supernatant (Fig. 1C).

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.

To understand UPR^mt regulation in C. elegans, we sought to determine the localization of ATFS-1 in the absence of UPR^mt-activation. We were unable to detect endogenous ATFS-1 or the ATFS-1::GFP fusion protein using ATFS-1 or GFP-specific antibodies (Figs. 1D & S2). Additionally, atfs-1_pr::gfp worms expressed GFP strongly in all cells indicative of an active promoter, while the ATFS-1::GFP fusion protein was nearly undetectable (fig. S3A). These data suggest that ATFS-1 is rapidly degraded.

We hypothesized that ATFS-1 was degraded by a mitochondrial matrix protease such as the caseinolytic peptidase ClpP or Lon (7). Animals fed lon(RNAi), but not clpp(RNAi) accumulated endogenous ATFS-1 as well as ATFS-1::GFP (Figs. 1D, S3B & S3C). ATFS-1 was absent in lysates from atfs-1(tm4525) worms (Figs. 1D & S3D), which were unable to activate the UPR^mt (fig. S4). lon(RNAi) did not affect atfs-1 transcription (fig. S5A). Furthermore, in lon(RNAi)-treated worms, ATFS-1 co-fractionated with a known mitochondrial protein (Figs. 1E & S3E). Unlike spg-7(RNAi), which impairs a mitochondrial protease required for electron transport chain (ETC) quality control and mitochondrial ribosome biogenesis (8), lon(RNAi) did not activate the UPR^mt transcriptional reporter hsp-60_pr::gfp or impair worm development (5) (Fig. 1F). Therefore, in the absence of UPR^mt-activation, ATFS-1 is imported into mitochondria and degraded.

During UPR^mt activation, ATFS-1::GFP accumulated in nuclei (Fig. 2A) (5). The predominant form of ATFS-1 that accumulated during spg-7(RNAi) or ethidium bromide (EtBr) treatment was of a higher molecular weight than the form detected in mitochondria of worms raised on lon(RNAi) and was enriched in the postmitochondrial supernatant (Figs. 2B & S4C) suggesting that during UPR^mt activation, a percentage of ATFS-1 remains in the cytosol, thus maintaining its MTS.

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.

Mitochondrial toxins such as paraquat, which activated the UPR^mt (Fig. 3A), are known to impair mitochondrial import causing the accumulation of MTS containing proteins in the cytosol (9). To determine if a general impairment of import occurs during UPR^mt activation, we generated a transgenic strain expressing GFP with a MTS (GFP^mt) (10, 11). Because steady-state detection of unprocessed MTS containing proteins is very difficult (12), we expressed GFP^mt via the inducible hsp-16 promoter (fig. S6). Only in the presence of UPR^mt-activating stress was unprocessed GFP^mt detected in the post-mitochondrial supernatant consistent with impaired import (Fig. 2C). Similarly, when ATFS-1 was expressed via the hsp-16 promoter, unprocessed ATFS-1 was only detectable in the postmitochondrial supernatant during UPR^mt-activating stress (Figs. 2D & S6). Import was not completely blocked during UPR^mt activation because the processed forms of ATFS-1 (revealed by lon(RNAi)) and GFP^mt were detected in mitochondria (Figs. 2C & 2D).

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.

Import into the matrix requires the TOM complex, the TIM23 complex, the ETC and the matrix-localized molecular chaperone mtHsp70 (1). tim-23(RNAi) caused ATFS-1::GFP to accumulate within nuclei (Fig. 2A) and strongly induced hsp-60_pr::gfp expression (Figs. 3A & S7). Furthermore, impairment of mtHsp70 (3) or the ETC by the isp-1(qm150) mutation (13) or cco-1(RNAi) also activated the UPR^mt suggesting that ATFS-1 responds to mitochondrial protein import perturbations (Fig. 3A).

We next considered how HAF-1, the previously identified UPR^mt regulator (5), affected ATFS-1. As expected, haf-1(ok705) worms were unable to induce hsp-60_pr::gfp expression caused by the clk-1(qm30) mutation (13) (Fig. 3B) or when raised on 30 μg/ml EtBr (Fig. 3A). However, hsp-60_pr::gfp induction caused by mitochondrial stresses that arrest worm development such as 100 μg/ml EtBr or spg-7(RNAi) treatment did not require haf-1 (fig. S8). Additionally, UPR^mt activation caused by conditions that directly inhibited mitochondrial import such as tomm-40(RNAi) (fig. S8A), tim-23(RNAi), cco-1(RNAi) or paraquat (9), also did not require haf-1 (Fig. 3A), suggesting that HAF-1 affects UPR^mt signaling by modulating the mitochondrial import of ATFS-1. During mitochondrial stress, steady state measurements indicated that more ATFS-1 accumulated within mitochondria of haf-1(ok705) worms as revealed by lon(RNAi) (Figs. 3B & S6B). Thus, in the absence of haf-1, ATFS-1 partitions to mitochondria during stress reducing UPR^mt activation.

To more directly examine the effect of haf-1(ok705) on ATFS-1 mitochondrial import efficiency, ATFS-1 was expressed from the inducible hsp-16 promoter (fig. S6). As discussed above, unprocessed ATFS-1 accumulated in the postmitochondrial supernatant during UPR^mt-activating stress. However, in haf-1(ok705) worms, much less ATFS-1 was detected in the cytosol (Fig. 2D). Similarly, the slowed import of GFP^mt during mitochondrial stress was not observed in haf-1(ok705) animals (Fig. 2C) suggesting that HAF-1 is a general attenuator of mitochondrial protein import during stress and is probably how HAF-1 modulates UPR^mt signaling.

To determine if prevention of ATFS-1 import into mitochondria is sufficient to cause ATFS-1 nuclear accumulation and UPR^mt activation, we generated a series of transgenic lines that expressed wild-type ATFS-1^{(full-length (FL))}, ATFS-1^Δ1-32.myc, an engineered variant of ATFS-1 unable to be imported into mitochondria, and ATFS-1^{Δ1-32.mycΔNLS} which lacked the NLS (fig. S9). Removal of the MTS was sufficient to cause nuclear accumulation of ATFS-1^Δ1-32.myc::GFP (Fig. 2A) and expression of ATFS-1^Δ1-32.myc caused constitutive expression of hsp-60_pr::gfp indicating that impaired mitochondrial import of ATFS-1 is sufficient for UPR^mt activation. Activation of hsp-60_pr::gfp by ATFS-1^Δ1-32.myc did not require haf-1 (Fig. 3C) further supporting a role for HAF-1 in mitochondrial import regulation. Mutating the NLS in ATFS-1 lacking the MTS prevented hsp-60_pr::gfp expression indicating that when ATFS-1 cannot be imported into mitochondria, ATFS-1 requires the NLS for UPR^mt activation (Figs. 3C & 3D).

To examine the physiological role of ATFS-1 during mitochondrial dysfunction, wild-type, clk-1(qm30) or isp-1(qm150) (13) worms were raised on control or atfs-1(RNAi). While unstressed worms were unaffected by atfs-1(RNAi), the mitochondrial stressed worms were unable to develop (Figs. 4A & S13A). Because ATFS-1 is regulated by mitochondrial import efficiency, a process linked to mitochondrial function, we sought to identify the entire ATFS-1 mediated response. Transcripts from wild-type and atfs-1(tm4525) worms raised in the presence and absence of stress were compared. A broad transcriptional response totaling 685 genes was induced during mitochondrial stress (Fig. 4B & Table S2), of which 391 required atfs-1 (Table S3).

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).

Included in the ATFS-1 program were many genes with roles in protecting against mitochondrial dysfunction (Fig. 4C) including the mitochondrial chaperones dnj-10 (14) (Fig. 4D) and hsp-60 (Fig. S13B). Numerous components involved in reactive oxygen species detoxification required ATFS-1 for induction during stress including the transcription factor skn-1 (15) (Fig. 4E). Several glycolysis genes including gpd-2 (glyceraldehyde-3-phosphate dehydrogenase) were also induced (Fig. 4F) suggesting the UPR^mt may contribute to a shift in ATP production from respiration to glycolysis. ATFS-1 was also required for the induction of tim-23 and tim-17 (Figs. 4G & S13C); core components of the TIM23 complex (1).

The presence of a NLS and a MTS in a single transcriptional activator allows the cell to monitor global mitochondrial import efficiency and determine the level of mitochondrial dysfunction. If mitochondria are functioning properly the constitutively synthesized ATFS-1 partitions into mitochondria where it is degraded. As mitochondrial dysfunction increases, mitochondrial import efficiency is reduced favoring translocation of ATFS-1 to the nucleus. Thus, mitochondrial homeostasis is maintained by the stress-dependent partitioning of a transcriptional activator between an inactive state in mitochondria and an active state in the nucleus.