UPRmt scales mitochondrial network expansion with protein synthesis via mitochondrial import

As organisms develop, individual cells generate mitochondria to fulfill physiologic requirements. However, it remains unknown how mitochondrial network expansion is scaled to cell growth and impacted by environmental cues. The mitochondrial unfolded protein response (UPRmt) is a signaling pathway mediated by the transcription factor ATFS-1 which harbors a mitochondrial targeting sequence (MTS)1. Here, we demonstrate that ATFS-1 mediates an adaptable mitochondrial expansion program that is active throughout normal development. Developmental mitochondrial network expansion required the relatively inefficient MTS2 in ATFS-1, which allowed the transcription factor to be responsive to parameters that impact protein import capacity of the entire mitochondrial network. Increasing the strength of the ATFS-1 MTS impaired UPRmt activity throughout development due to increased accumulation within mitochondria. The insulin-like signaling-TORC13 and AMPK pathways affected UPRmt activation4,5 in a manner that correlated with protein synthesis. Manipulation to increase protein synthesis caused UPRmt activation. Alternatively, S6 kinase inhibition had the opposite effect due to increased mitochondrial accumulation of ATFS-1. However, ATFS-1 with a dysfunctional MTS6 constitutively increased UPRmt activity independent of TORC1 function. Lastly, expression of a single protein with a strong MTS, was sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent manner. We propose that mitochondrial network expansion during development is an emergent property of the synthesis of highly expressed mitochondrial proteins that exclude ATFS-1 from mitochondrial import, causing UPRmt activation. Mitochondrial network expansion is attenuated once ATFS-1 can be imported.


Abstract 25
As organisms develop, individual cells generate mitochondria to fulfill physiologic 26 requirements. However, it remains unknown how mitochondrial network expansion is scaled 27 to cell growth and impacted by environmental cues. The mitochondrial unfolded protein 28 response (UPR mt ) is a signaling pathway mediated by the transcription factor ATFS-1 which 29 harbors a mitochondrial targeting sequence (MTS) 1 . Here, we demonstrate that ATFS-1 30 mediates an adaptable mitochondrial expansion program that is active throughout normal 31 development. Developmental mitochondrial network expansion required the relatively 32 inefficient MTS 2 in ATFS-1, which allowed the transcription factor to be responsive to 33 parameters that impact protein import capacity of the entire mitochondrial network. Increasing 34 the strength of the ATFS-1 MTS impaired UPR mt activity throughout development due to 35 increased accumulation within mitochondria. The insulin-like signaling-TORC1 3 and AMPK 36 pathways affected UPR mt activation 4,5 in a manner that correlated with protein synthesis. 37 Manipulation to increase protein synthesis caused UPR mt activation. Alternatively, S6 kinase 38 inhibition had the opposite effect due to increased mitochondrial accumulation of ATFS-1. 39 However, ATFS-1 with a dysfunctional MTS 6 constitutively increased UPR mt activity 40 independent of TORC1 function. Lastly, expression of a single protein with a strong MTS, 41 was sufficient to expand the muscle cell mitochondrial network in an ATFS-1-dependent 42 manner. We propose that mitochondrial network expansion during development is an 43 emergent property of the synthesis of highly expressed mitochondrial proteins that exclude 44 ATFS-1 from mitochondrial import, causing UPR mt activation. Mitochondrial network 45 expansion is attenuated once ATFS-1 can be imported. The UPR mt is a mitochondrial-to-nuclear signal transduction pathway regulated by the 50 transcription factor ATFS-1 that is required for development and longevity during 51 mitochondrial dysfunction 1,7,8 . Because ATFS-1 harbors a MTS and a nuclear localization 52 sequence (NLS), its transcription activity is regulated by subcellular localization. If ATFS-1 is 53 imported into mitochondria, it is degraded by the protease LONP-1 1 (Fig. 1a). However, if a 54 percentage of ATFS-1 fails to be imported into mitochondria, it traffics to the nucleus to 55 activate a transcriptional response that includes mitochondrial chaperones 9,10 . Perturbations 56 to OXPHOS or mitochondrial proteostasis activate the UPR mt as both processes are required 57 for mitochondrial protein import 11 . 58 59 A role for ATFS-1 in mitochondrial network maintenance and expansion 60 We previously found that OXPHOS dysfunction due to deleterious mtDNA heteroplasmy 61 caused an atfs-1-dependent expansion of the mitochondrial network that was observed only 62 when mitophagy was impaired 12 . Similarly, OXPHOS dysfunction caused by mutations in the 63 ubiquinone biogenesis gene clk-1, induced the UPR mt and lead to an increase in mtDNA 64 (Extended Data Fig. 1a) suggesting a role for the UPR mt in mitochondrial biogenesis or network 65 expansion. 66 atfs-1(et18) worms constitutively activate the UPR mt due to an amino acid substitution 67 in the MTS which impairs import into mitochondria even in the absence of mitochondrial 68 stress 6 . Impressively, atfs-1(et18) worms harbored more mtDNAs relative to wildtype 69 worms (Fig. 1b), suggesting that UPR mt activation is sufficient to expand the mitochondrial 70 network. Conversely, worms lacking the entire atfs-1 open reading frame (atfs-1(null)) 13 71 had reduced mtDNAs (Fig. 1c). Moreover, TMRE staining indicated that atfs-1(null) or 72 network 1 . ATFS-1 is predicted to have a relatively weak, or inefficient, MTS compared to 119 other mitochondrial-targeted proteins such as mitochondrial chaperones and OXPHOS 120 components 15,16 (Fig. 3a). To compare the MTS strength of the OXPHOS protein ATP 121 synthase subunit 9 (Su9) to ATFS-1, the amino-terminus of each was fused to GFP and 122 expressed in HEK293T cells. As expected, both GFP-fusion proteins accumulated within 123 mitochondria, but unlike Su9 (1-69) ::GFP, ATFS-1 (1-100) ::GFP fluorescence also 124 accumulated within the cytosol, but to a lesser extent than that of ATFS-1 et18(1-100) ::GFP 125 ( Fig. 3b). Additionally, import of ATFS-1 (1-100) ::GFP was limited compared to Su9 (1-126 69) ::GFP in an in vitro import assay (Fig. 3d) consistent with ATFS-1 harboring a weak 127

Discussion 234
In summary, we have found that ATFS-1 regulates a mitochondrial expansion program 235 that is active throughout normal development. Developmental mitochondrial expansion 236 required the inefficient MTS of ATFS-1 and TORC1 activity suggesting an interplay 237 between protein synthesis, mitochondrial protein import capacity, and nuclear activity of 238 ATFS-1. Consistent with these findings, OXPHOS transcripts are among the most highly 239 suggesting import or intra-mitochondrial protein processing can be overwhelmed during 248 normal cell growth. We propose a model where the high levels of mitochondrial protein 249 synthesis that occurs during development drives mitochondrial network expansion by 250 excluding a percentage of ATFS-1 from mitochondrial import. And, network expansion 251 continues until import is sufficient to import ATFS-1 and terminate the UPR mt . These 252 findings are conceptually similar to the endoplasmic reticulum expansion that occurs in 253 response to increased protein flux via the UPR ER , which is regulated by IRE1 and XBP1 29 . 254 We propose that as a function of the mitochondrial import flux or mitochondrial protein 255 processing, ATFS-1 scales mitochondrial network expansion with cell growth. 256

Protein analysis and antibodies 284
Synchronized worms were raised on plates with control(RNAi) or lonp-1(RNAi) to the L4 285 stage prior to harvesting. Whole worm lysate preparation was previously described 30 . 286 Antibodies against α-tubulin were purchased from Calbiochem (CP06), GFP and for 287 NDUFS3 from Abcam (ab6556 and ab14711 respectively). Antibodies for ATFS-1 were 288 previously described 1 . Immunoblots were visualized using ChemiDoc XRS+ system (Bio-289 Rad). All western blot experiments were performed at least three times.

Oxygen Consumption 317
Oxygen consumption was measured using a Seahorse XFe96 Analyzer at 25°C similar 318 to that described previously 37 . In brief, L4 worms were transferred onto empty plates and 319 allowed to completely digest the remaining bacteria for 1 hour, after which 10 worms were 320 transferred into each well of a 96-well microplate containing 180 µl M9 buffer. Basal 321 respiration was measured for a total of 30 minutes, in 6 minute intervals that included a 2 322 minute mix, a 2 minute time delay and a 2 minute measurement. To measure respiratory 323 capacity, 15 µM FCCP was injected, the OCR (oxygen consumption rate) reading was 324 allowed to stabilize for 6 minutes then measured for five consecutive intervals. The cDNA libraries were run on an Illumina HiSEq2000 instrument with single-read 50-336 bp (SR50). RNA reads were then aligned to WBcel235/ce11 reference genome and 337 differential gene expression analysis was performed with edgeR 38 . Differences in gene 338 expression between atfs-1(et18) and atfs-1(null) compared to wildtype are listed in 339 Supplementary Tables 6 and 7 respectively. 340 341

Analysis of worm development 342
Worms were synchronized via bleaching and allowed to develop on HT115 bacteria 343 plates for 3 days at 20°C. Developmental stage was quantified as a percentage of the 344 total number of animals. Each experiment was preformed three times. For the comparison 345 of wildtype and atfs-1(null) worms; N=162 (wildtype), and 282 (atfs-1(null)). For the 346 comparison of wildtype to atfs-1 R/R worms; N=158 (wildtype) and N=256 (atfs-1 R/R ). Uranyl acetate. Sections were examined using a CM10 TEM with 100Kv accelerating 387 voltage, and images were captured using a Gatan TEM CCD camera. 388 389

Ribosome profiling data analysis 390
Ribosome profiling sequencing data was downloaded from the NCBI Sequence Read 391 Archive (SRA) (http://www.ncbi.nlm.nih.gov/sra/) under accession number SRA055804. 392 Data was analyzed as previously described 25 . Data analysis was done with the help of 393 Unix-based software tools. First, the quality of raw sequencing reads was determined by 394 FastQC 42 . Reads were then filtered according to quality via FASTQ for a mean PHRED 395 quality score above 30 43 . Filtered reads were mapped to the C. elegans reference 396 genome (Wormbase WS275) using BWA (version 0.7.5) and SAM files were converted 397 into BAM files by SAMtools (version 0.1.19). Coverage data for specific genes (including 398 5'UTR, exons and 3'UTR) were calculated by SAMtools and coverage data for each gene 399 was plotted using R 44 . Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation.  e. TMRE staining of wildtype worms raised on control or atfs-1(RNAi) and atfs-1(null) worms. Skeleton-like binary backbone is presented (bottom). Scale bar 10µm.
j. Quantification of mtDNA in wildtype and atfs-1 R/R worms as determined by qPCR. N=3.