PERK recruits E-Syt1 at ER–mitochondria contacts for mitochondrial lipid transport and respiration

ER–mitochondria contact sites (EMCS) regulate non-vesicular phospholipid transport between these organelles, but the molecular entities involved in lipid translocation remain undefined. Sassano et al. reveal that EMCS-associated PERK recruits the lipid transfer protein E-Syt1 at EMCS facilitating lipid transport to sustain mitochondrial homeostasis and respiration.


Introduction
The ER-mitochondria contact sites (EMCS) are subdomains of smooth ER in close apposition (usually in the range of 10-80 nm) to the mitochondrial outer membrane, which are maintained by dedicated tether, spacer, and lipid transfer proteins (LTPs; Scorrano et al., 2019). EMCS, also termed mitochondriaassociated ER membranes (MAMs), are essential regulators of Ca 2+ , metabolites, and phospholipid (PL) transfer between these organelles (Prinz et al., 2020;Vance, 2014). A long appreciated, functional relationship exists between the architectural integrity of EMCS and mitochondrial lipid trafficking (Vance, 1991). Indeed, the lipid composition and structural organization of mitochondrial membranes, depend in large part on the import of essential lipids and precursors from the ER (Ardail et al., 1991;Kornmann et al., 2009;Tamura et al., 2012;Voelker, 1984). While aberrant lipid trafficking between ER and mitochondria is emerging as a persuasive characteristic of aged cells and diseases like neurodegeneration and cancer (Doghman-Bouguerra and Lalli, 2019; Sassano et al., 2017;Wilson and Metzakopian, 2021), a precise understanding of the molecular components regulating this process is lacking. The expanding physiological roles of EMCS argue that their molecular composition and size are tightly regulated by dynamic recruitment of multifunctional proteins, tethers, and LTPs in order to tailor cellular responses to fluctuating nutritional and stress cues. Assorted ER-resident proteins organize in molecular complexes at the EMCS of mammalian cells (Scorrano et al., 2019). These include PERK (EIF2AK3) and IRE1α, two main effectors of the unfolded protein response (UPR; Fan and Simmen, 2019;Rainbolt et al., 2014;Ron and Walter, 2007;van Vliet and Agostinis, 2018). The UPR is a major transcriptionally regulated adaptive pathway, activated by loss of ER folding capacity following different cellular stresses, such as ER-Ca 2+ store depletion and glucose deprivation. This results in the accumulation of misfolded proteins in the ER lumen, a condition known as ER stress (Almanza et al., 2019;Ron and Walter, 2007). In particular, the PERK branch of the UPR temporarily halts protein synthesis by phosphorylating eIF2α, while simultaneously increasing the expression of the transcription factor ATF4, which regulates autophagy, amino acid metabolism, oxidative stress, and apoptosis, according to the intensity of ER stress (Harding et al., 2000;Hetz, 2012). Recent studies suggest that in different cellular contexts, PERK maintains mitochondrial homeostasis, mitochondria cristae junctions, and bioenergetics in response to ER stress, through transcriptional-dependent and independent mechanisms. While the molecular underpinning of the former has been confirmed by genetic and pharmacological approaches (Balsa et al., 2019;Chakraborty et al., 2022;Latorre-Muro et al., 2021;Lebeau et al., 2018;Muñoz et al., 2013;Raines et al., 2022), how PERK regulates mitochondrial homeostasis independent of its kinase activity remains elusive. Interestingly, following oxidative ER stress, PERK was shown to prime mitochondrial cell death by tightening the EMCS independent of its kinase activity (Verfaillie et al., 2012). Notwithstanding, whether and how EMCS-associated PERK regulates homeostatic lipid trafficking remains unexplored. This knowledge is particularly relevant considering that the pathogenetic role of PERK mutations in a spectrum of diseases, including the Wolcott-Rallison syndrome (WRS; Julier and Nicolino, 2010), is increasingly linked to mitochondrial dysfunctions and aberrant lipid metabolism (Julier and Nicolino, 2010;Oberhauser and Maechler, 2021), which cannot be explained uniquely by its canonical role in the UPR.
Here, we reveal that PERK promotes lipid transfer between the ER and mitochondria in unstressed cells. We found that this PERK function requires the recruitment of the lipid transfer protein Extended-Synaptotagmin 1 (E-Syt1) through its C2D-C2E domains at EMCS. Using E-Syt1 deletion mutants perturbing PERK-E-Syt1 interaction or ability of E-Syt1 to transport lipids, we portrayed the functional relevance of this axis to preserve mitochondrial lipid homeostasis and respiration.

Results
PERK regulates ER-mitochondria phospholipid transfer and mitochondrial lipid homeostasis Using conventional confocal microscopy, we reported that PERK regulates EMCS under conditions of oxidative ER stress in MEF cells (Verfaillie et al., 2012). Given the limit of the Z resolution of confocal microscopy, we first set out to obtain more accurate 3D images with superior Z-axis resolution of the effects of PERK on EMCS integrity in homeostatic conditions by using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM). For this analysis, we used HeLa cells stably transduced with a control shRNA (shCTR) or shRNA against PERK (shPERK; resulting in ∼60% reduction at the protein levels ( Fig. S1 A), thus expanding previous data in a different cellular model. 3D reconstitutions of FIB-SEM stacks were used to measure key structural parameters of EMCS-considered as distance equal or less than 25 nmnamely, mitochondrial surface in contact with the ER and EMCS size (Helle et al., 2013; Fig. 1 A, Video 1, Video 2, Video 3, and Video 4). Consistent with previous reports (Hirabayashi et al., 2017;Rizzuto et al., 1998), in shCTR cells, around 20% of mitochondrial surface was in contact with the ER, and this dropped to 10% in shPERK cells (Fig. 1 B). In PERK-silenced cells, the total ER-mitochondria contact area per mitochondria was reduced ( Fig. 1 C), while overall mitochondrial morphological parameters ( Fig. 1 D and Fig. S1, B and C) and mass (Fig. S1 D) were unaffected. Thus, PERK contributes to maintain EMCS integrity in unstressed cells.
EMCS constitute the molecular platform for the transport of phosphatidylcholine (PC), phosphatidylinositol (PI), and phosphatidylserine (PS), which are the most abundant PLs in cellular membranes, from the ER where they are synthesized to the mitochondria (Kannan et al., 2017;Tamura et al., 2012;Vance, 2020). Once shuttled to the mitochondria, PS is rapidly converted to phosphatidylethanolamine (PE) by decarboxylation catalyzed by the inner membrane PS decarboxylase (PSD; Vance, 1990).
To decipher the role of PERK in this process, we loaded HeLa cells with 18:1-06:0 N-[7-Nitrobenz-2-oxa-1,3-diazol-4-yl] phosphatidylserine (NBD-PS), a fluorescent analog of PS which can be used to monitor the traffic from ER membranes and subsequently to mitochondrial membranes, where it is rapidly converted into PE (Helle et al., 2013). Live-cell imaging showed that in shCTR cells, NBD-PS redistributed from a main cytoplasmic/ER membrane localization to the mitochondrial network ( Fig. 1, E and F) within 30 min, as previously reported (Hailey et al., 2010). In contrast, in shPERK cells, redistribution of NDB-PS to the mitochondria was significantly reduced (Fig. 1, E and F).
We then tested the functional role of PERK in PL trafficking in human fibroblasts derived from a healthy donor and a 2.5-mo-old girl presenting with Wolcott-Rallison Syndrome (WRS) clinically associated with neonatal diabetes and liver dysfunction, caused by a homozygous p.W681X/p.W681X nonsense mutation in EIF2AK3 (PERK). This resulted in the expression of a PERK truncated variant lacking a large portion of the C-terminal PERK kinase domain (Fig. S1 E). Rapidly after the addition of the fluorescent PS analog, healthy fibroblasts (CTR) showed a clear pattern of mitochondrially redistributed NBD-PS (Fig. 1,G and H). In contrast, in WRS fibroblasts, mitochondriaassociated PS signal was significantly diminished (Fig. 1,G and H). Albeit WRS fibroblasts exhibited signs of an altered mitochondrial network, as observed in other pathological conditions caused by chronic loss of PERK signal (Almeida et al., 2022;Lebeau et al., 2018), these results support a role of PERK in ER-mitochondria PL transport.
We next analyzed the conversion of exogenously added NBD-PS to NBD-PE in shCTR and shPERK HeLa cells by onedimensional thin-layer chromatography (TLC), allowing the separation of PS and PE based on their different polarity (Fig. 2 A;Hölzl and Dörmann, 2021;Watanabe et al., 2020). When calculating the ratio NBD-PE/(NBD-PS + NBD-PE), we found that in PERK silenced cells NBD-PS to NBD-PE conversion was significantly reduced as compared to shCTR cells (Fig. 2 B), further suggesting a role for PERK in ER-mitochondria PS transport.
We then asked whether PERK silencing altered the content of several mitochondrial PLs and/or their precursors, which are regulated by the ER-mitochondria shuttle. To this end, we performed quantitative lipidomics from purified mitochondrial fractions of shCRT and shPERK HeLa cells (Fig. S1 F) and wildtype (PERK +/+ ) and PERK knock out (PERK −/− ) murine embryonic fibroblast (MEFs; Fig. S1 G). The abundance of PC, PI, PE (PS Figure 1. PERK regulates the integrity of the EMCS and phospholipid trafficking. (A) Representative electron micrographs and 3D segmentation reconstructed from FIB-SEM image stacks (red = mitochondria, green = ER/EMCS, blue = nucleus) of shCTR and shPERK HeLa cells transiently transfected with is rapidly transformed into PE in the mitochondria), and phosphatidylglycerol (PG; Fig. S1 H) was attenuated in the purified mitochondrial fractions of PERK silenced cells (Fig. 2 C) or PERK −/− cells (Fig. 2 D) as compared to their corresponding PERK proficient counterparts. However, overall mitochondrial PL abundance remained unchanged (Fig. 2,E and F) hinting that other PLs may compensate for the observed reduction in PC, PI, and PE. In line with this, we found an increased trend in the mitochondrial content of diacylglycerol (DG) of shPERK compared to their shCTR ( Fig. S1 I). Notably, the overall cellular content of PC, PE, PI, PG, and PS was not grossly changed (Fig. S1 J). Together, these data disclose that PERK deficiency impairs the ER-mitochondrial transport of key phospholipids.
PERK recruits the lipid transfer protein EXTENDED SYNAPTOTAGMIN-1 at the EMCS We next addressed the mechanisms through which PERK regulates mitochondrial lipid homeostasis. PERK silencing did not alter the expression of the mitochondrial PSD nor of the ERassociated PS synthase1/2 (PSS1, PSS2; Fig. S2 A). Our recent PERK interactome studies (Sassano et al., 2021;van Vliet et al., 2017) revealed E-Syt1 as a potential interactor of PERK. E-Syt1 is a member of the conserved E-Syt family of proteins, which in mammals also includes E-Syt2 and E-Syt3. E-Syts are ERanchored LTPs with a known role in favoring ER-PM tethering (Idevall-Hagren et al., 2015;Kang et al., 2019;Saheki and De Camilli, 2017). We focused on E-Syt1 as a plausible functional PERK interactor given that E-Syt1 is a LTP for which a limited number of interacting partners with ER-PM tethering functions are known (Chang et al., 2013;Kang et al., 2019). Moreover, the possibility that lipid homeostasis could be regulated by a PERK-E-Syt1 interaction at EMCS is unexplored.
We first validated that PERK and E-Syt1 co-immunoprecipitated in HEK-293T cells, by co-transfecting myc-tagged PERK or its kinase-dead mutant (PERK K618A ) with eGFP-tagged E-Syt1, to control for equal protein expression ( Fig. 3 A and Fig. S2 B). Because of the close molecular weight of eGFP-E-Syt1 (146 kD) and myc-tagged PERK (126 kD), both proteins run at a similar height in the gels but were distinguishable as independent bands (Fig. 3 A). A residual GFP-E-Syt1 signal was still visible as upper band in the anti-myc panel, due to spectral bleed-through in our fluorescence-based detection system. PERK and even more the PERK K618A mutant although not significantly (Fig. 3 B) coimmunoprecipitated with eGFP-E-Syt1 (Fig. 3 A). In contrast, the integral ER protein calnexin (CNX) was detected in the total lysates but was absent in the co-IP, further indicating the specificity of the PERK-E-Syt1 interaction. These results were further confirmed in other co-IP settings using cells expressing eGFPtagged E-Syt1 followed by GFP-pull down of endogenous PERK (Fig. 3 C).
Thus, PERK independent of its kinase activity interacts, either directly or indirectly, with E-Syt1, confirming our previous Bio-ID data (Sassano et al., 2021).
E-Syt1 has been involved in ER-PM contact formation upon rise in intracellular Ca 2+ (Idevall-Hagren et al., 2015). We then utilized immunogold electron microscopy (IEM) to visualize whether a fraction of eGFP-E-Syt1 could be found in close proximity with the mitochondria, in resting conditions. Morphological analysis by IEM showed that in HeLa cells around 10% of the total amount of eGFP-E-Syt1 was located at EMCS, a fraction similar to the amount of eGFP-E-Syt1 present at the ER-PM contact sites (Fig. 3, D and E). We then isolated crude mitochondrial fractions (consisting of mitochondria and ERassociated membranes), pure mitochondrial fractions and the EMCS fraction (or MAMs) from shCTR and shPERK HeLa cells (Fig. 3 F) and PERK +/+ and PERK −/− MEFs (Fig. S2 C) to analyze the presence of E-Syt1 in these subcellular compartments by immunoblotting. E-Syt1 was found accumulated in the EMCS fraction-while being absent from purified mitochondria as expected-along with PERK, and the known MAM markers Calnexin (CNX) and IP3R3 (Fig. 3 F and Fig. S2 C). At equal EMCS proteins amount, MAM-associated E-Syt1 levels were reduced in absence of PERK (Fig. 3, F and G and Fig. S2, C and D) while this was not observed in total lysates (Fig. 3,F and H;and Fig. S2,C and E), suggesting that PERK may favor E-Syt1 redistribution to EMCS. To corroborate this hypothesis, we then used live-cell imaging in PERK-proficient or depleted Hela cells expressing eGFP-E-Syt1 and visualized mitochondrial network with Mito-Tracker Far Red. In all conditions, eGFP-E-Syt1 signal was distributed as expected within the entire ER network (Giordano et al., 2013). However, while in PERK-proficient cells, a fraction of eGFP-E-Syt1 signal was intermingled with the mitochondrial network; this was reduced in PERK-depleted cells (Fig. 3, J and K). This effect was PERK-specific and independent of its basal kinase activity since reintroduction of the PERKkinase dead mutant in these cells (Fig. 3 I) rescued this defect to an extent comparable to that of PERK-proficient cells (Fig. 3, J and K). These results suggest that PERK, independently of its HRP-KDEL-myc. EMCS are indicated by dashed light blue lines (≤25 nm distance between membranes). Scale bar, 1 µm. (B) Percentage of contact surface normalized on the total mitochondrial surface/mitochondria. The values plotted are the mean ± SEM from two biological replicates (n = 11) analyzed using unpaired Student's t test. (C) Total ER-mitochondria contact area/mitochondria. The values plotted are the mean ± SEM from two biological replicates (n = 11) analyzed using unpaired Student's t test. (D) Average of mitochondrial surface area/volume. The values plotted are the mean ± SEM from two biological replicates (n = 11) analyzed using unpaired Student's t test. (E) Representative images of NBD-PS co-stained with MitoTracker Far Red in shCTR and shPERK HeLa cells. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (F) NBD-PS and MitoTracker Far Red colocalization analysis of shCTR and shPERK HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates (n = 37 and n = 25 for shCTR and shPERK, respectively) analyzed using unpaired Student's t test. (G) Representative images of NBD-PS co-stained with MitoTracker Far Red in CTR and p.W681X PERK mutant human fibroblasts. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (H) NBD-PS and MitoTracker Far Red colocalization analysis of CTR and p.W681X human fibroblasts (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates (n = 27 and n = 28 for CTR and p.W681X, respectively) analyzed using unpaired Student's t test. *,P < 0.05; ****,P < 0.0001; and NS = not significant. See also Video 1, Video 2, Video 3 and Video 4. kinase activity, recruits a fraction of ER-associated E-Syt1 at the intersection between the ER and the mitochondria.
We then investigated whether reducing E-Syt1 expression affected EMCS integrity and mitochondrial lipid composition.
Using orthogonal readouts, we found that in contrast to PERK depletion, reducing E-Syt1 expression did not affect the average number of EMCS per cell (as measured by the PLA assay; Fig. S2, F and I) nor the mitochondrial surface area engaged into EMCS (defined as membrane appositions between the two organelles with <30 nm distance, as measured by HRP-KDEL-myc EM analysis; Fig. S2, J and K). Considering that differences in the number or in the morphology of mitochondria may affect EM analysis of EMCS, we measured the average number of mitochondria per cell and their morphology. Both parameters were unaltered in E-Syt1 silenced cells (Fig. S2, J and K).
Consistent with the observations that optimal Ca 2+ transfer between the ER and mitochondria occurs through EMCS with a width that lies between 15 and 20 nm (Csordás et al., 2006;Lim et al., 2021), silencing E-Syt1 expression did not elicit alterations in mitochondrial Ca 2+ transfer (as measured by the mitochondria-targeted protein Aequorin) in response to IP3 generating stimulus ATP ( A PERK-E-Syt1 axis mediates phospholipid trafficking at EMCS We then set out to gain more insights into the functional effects of E-Syt1-PERK interaction in lipid transfer. E-Syt1 harbors an amino-terminal ER-membrane anchor, a synaptotagmin-like mitochondrial-lipid-binding protein domain (SMP), and five C2 domains: C2A, C2B, C2C, C2D, and C2E, with the C2D-C2E only found in E-Syt1 (Saheki and De Camilli, 2017). Following intracellular Ca 2+ rise, positively charged C2C domain of E-Syt1 binds PM-rich PI(4,5)P2, favoring ER-PM tethering (Bian et al., 2018;Idevall-Hagren et al., 2015). SMP is a member of the tubular lipid-binding protein (TULIP) superfamily typically present in proteins associating with membrane contact sites (MCS) with roles in transporting lipids between the ER and other organelles (Prinz et al., 2020;Wong and Levine, 2017).
Our previous Bio-ID interactome analysis did not reveal E-Syt2 as a proximity interactor of PERK (Sassano et al., 2021). However, because ubiquitously expressed E-Syt1 and E-Syt2 share similar structural domains, to further examine the specific role of E-Syt1 as PERK partner, we used E-Syt1/2 double knocked out (DKO) HeLa cells and reconstituted these with eGFP-tagged full length E-Syt1 (E-Syt1FL). eGFP pull-down experiments were performed from isolated crude mitochondrial (mito crude) fractions and showed that endogenous PERK was recovered in eGFP-E-Syt1 expressing DKO cells, but not in cells expressing GFP alone ( Fig. 4 A and Fig. S4 A). This suggests that PERK and E-Syt1 interact at the ER-mitochondria interfacing membranes. We then assessed which domain(s) of E-Syt1 was responsible for interaction with PERK. To this end, along with E-Syt1FL, we expressed various eGFP-tagged E-Syt1 deletion mutants, removing either the SMP domain (E-Syt1ΔSMP), the C2C, C2D, and C2E domains (E-Syt1ΔCDE) or the C2D and C2E domains (E-Syt1ΔDE; Fig. 4 B). All these mutants were expressed in DKO cells at a similar extent and comparable to E-Syt1 endogenous levels (Fig. S4, B and C). Co-IP analysis of crude mitochondrial fraction of DKO cells ( We then hypothesized that DE-mediated PERK-E-Syt1 association is required for E-Syt1 localization at the EMCS. DKO cells reconstituted with eGFP-tagged E-Syt1FL or E-Syt1ΔSMP displayed a similar fraction of the eGFP signal intertwined with the mitochondrial network, whereas this colocalization was significantly tempered by PERK-interaction defective E-Syt1ΔDE mutant (Fig. 4, G and H). Congruently, the co-expression of eGFP-tagged E-Syt1FL or E-Syt1ΔDE mutant with STIM1-mCherry (which colocalizes with E-Syt1 in resting conditions and following ER-Ca 2+ store depletion [Kang et al., 2019]), showed that the PERK-interaction defective mutant displayed an increased ER localization compared to E-Syt1FL (  The values plotted are the mean ± SEM from three biological replicates analyzed using unpaired Student's t test. (C) Abundance of PC, PI, PE from purified mitochondrial fractions of shCTR and shPERK HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from 4 biological replicates analyzed using one sample t test. (D) Abundance of PC, PI, PE from purified mitochondrial fractions of PERK +/+ and PERK −/− MEF cells, relative to control condition (PERK +/+ ). The values plotted are the mean ± SEM from four biological replicates analyzed using one sample t test. (E) Abundance of total mitochondrial phospholipids from purified mitochondrial fractions of shCTR and shPERK HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from four biological replicates analyzed using one sample t test. (F) Abundance of total mitochondrial phospholipids from purified mitochondrial fractions of PERK +/+ and PERK −/− MEF cells, relative to control condition (PERK +/+ ). The values plotted are the mean ± SEM from four biological replicates analyzed using one sample t test. *, P < 0.05; **, P < 0.01; and NS = not significant. Source data are available for this figure: SourceData F2. The PERK-E-SYT1 axis maintains mitochondrial respiration under steady-state Alterations of mitochondrial PL composition, and in particular PE abundance, impair formation and/or membrane integration of respiratory chain complexes (Baker et al., 2016;Basu Ball et al., 2018;Joshi et al., 2012;Tasseva et al., 2013) and could have a significant impact on mitochondrial respiration (Mejia and Hatch, 2016). The PERK-eIF2α-ATF4 signal has been shown to regulate mitochondrial metabolism in response to ER stress, but the impact of PERK-E-Syt1 axis on basal oxygen consumption rate (OCR) is unknown.
To address this question and because HeLa cells produce large part of their ATP through the degradation of glucose (Depaoli et al., 2018), we used conditions that favor oxidative phosphorylation and halt glycolysis. To this end, we replaced the glucose-containing medium with galactose just before measuring OCR. This short-term exposure to galactose did not elicit signs of ER stress (data not shown), which are commonly observed after prolonged glucose deprivation (Iurlaro et al., 2017).
PERK-depleted HeLa cells displayed reduced basal, maximal (uncoupled) respiration, and lower ATP production, which were largely corrected by the re-expression of PERK kinase-dead mutant (Fig. 6, A and B). Consistent with this, WRS-fibroblasts also showed a severely impaired OCR (Fig. 6, C and D) in comparison with healthy fibroblasts whose ability to respire was unaffected by the chemical inhibition of PERK (PKI; Fig. 4 and Fig. 6, C-E).
To evaluate the impact of the PERK-E-Syt1 interaction on oxidative phosphorylation and couple it to the effects observed in PS trafficking (Fig. 5, A-F), we then measured OCR in DKO cells and upon re-expression of E-Syt1FL, E-Syt1ΔDE, or E-Syt1ΔSMP mutants (Fig. S5, C and D). Compared to WT cells, DKO cells exhibited a significantly impaired basal and maximal respiration, and re-expression of E-Syt1FL partially rescued OCR in these cells (Fig. 6, F and G). The observation that E-Syt1 reexpression in DKO HeLa cells does not fully recover the rate of mitochondrial respiration observed in WT cells suggests a possible contribution of E-Syt2 in this process. Notably, expression of the PERK-interaction defective mutant E-Syt1ΔDE or the E-Syt1ΔSMP, which can still bind PERK but is devoid of lipid transfer function, failed to do so (Fig. 6, F and G).
Thus, perturbing the PERK-E-Syt1 interaction or removing the E-Syt1 domain responsible for PL transport (SMP) compromises mitochondrial respiration (Fig. 6 H).

Discussion
Our study portrays that PERK is a key component of the molecular machinery that maintains homeostatic ER-mitochondria transport of PLs. We further revealed that this function of PERK is favored by heterotypic interaction with the lipid transport protein E-Syt1 at EMCS. Functionally, within the PERK-E-Syt1 (PERKK618A) and with eGFP-empty vector or eGFP-tagged E-Syt1. Untransfected cells are shown as negative control. In the eGFP-E-Syt1 transfected cells, a residual GFP signal is still visible as upper band (blue arrow) above PERK (red arrow) in the anti-myc panel. (B) Quantification of the PERK-E-Syt1 interaction normalized on GFP-pulled down and relative to control condition (PERK-myc). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (C) Representative immunoblot for eGFP and PERK showing the interaction of PERK and E-Syt1 in HEK293-T cells transiently transfected with eGFP-empty vector or eGFP-tagged E-Syt1. Arrows indicate GFP signals for eGFP-empty vector and eGFP-E-Syt1 pulled down. (D) Representative electron micrograph of ultrathin cryosections of HeLa cells transfected with eGFP-E-Syt1 and immunogold stained with anti-GFP (15 nm gold particles) and anti-PDI (10 nm gold particles). Black arrow denotes E-Syt1 detection at the sites of juxtaposition between the ER and the mitochondria membranes, while PDI, a general ER marker, remains in the ER lumen. Scale bar, 500 nm. (E) Relative quantification of the cellular distribution of E-Syt1 in the ER, EMCS (MAMs), and plasma membrane (PM). The values plotted are the mean ± SEM (n = 16 cellular profiles). (F) Representative immunoblot for IP3R3, PERK, E-Syt1, CNX, VDAC1 and CYTC from total lysates, crude mitochondrial fraction (mito crude), purified mitochondrial fraction (mito pure) and MAM fraction of shCTR and shPERK HeLa cells. (G and H) Quantification of E-Syt1 level at MAMs (G) and total lysate (H) normalized on CNX levels and relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (I) Representative immunoblot for PERK in shCTR, shPERK, and shPERK + PERKK618A HeLa cells. ACTIN serves as loading control. (J) Representative images from eGFP-E-Syt1 transiently transfected and co-stained with MitoTracker Far Red in shCTR, shPERK and shPERK + PERKK618A HeLa cells. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (K) Colocalization analysis of E-Syt1 and MitoTracker Far Red in shCTR, shPERK and shPERK + PERKK618A HeLa cells (Manders M1 coefficient). The values plotted are the mean ± SEM from three biological replicates (n = 26, n = 26, and n = 25 for shCTR, shPERK, and shPERK + PERKK618A respectively) analyzed using one-way ANOVA, with Tukey's test for multiple comparisons. (L) Abundance of PC, PI, PE from purified mitochondrial fractions of shCTR and shE-Syt1 HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; and NS = not significant. Source data are available for this figure: SourceData F3.
axis, we showed that the expression of E-Syt1 mutants unable to bind PERK fails to rescue the defects in PL transport and mitochondrial respiration observed in E-Syt1/2 DKO cells, while expression of the full length E-Syt1 largely recovers these deficits.
This study reveals that E-Syt1 interacts with PERK through its unique C-terminal C2D-C2E domain and that a fraction of E-Syt1 is found at EMCS in resting cells. Single molecule studies (Bian et al., 2018) indicated that at low Ca 2+ concentration, the E-Syt1-C2E domain exhibits an intrinsically weak affinity for PM. This may suggest that in resting conditions E-Syt1 may participate in other MCS functions. However, since lipid binding through the SMP domain is modulated by Ca 2+ (Bian et al., 2018), it is possible, albeit speculative, that local concentration of Ca 2+ within the microdomains of EMCS (Csordás et al., 2010) may be sufficient to promote SMP PL binding. Alternatively, interaction with PERK may facilitate a conformational change in E-Syt1 releasing charge-based autoinhibitory interaction between the C2A and SMP domains, an interesting hypothesis requiring further structural analysis of PERK-E-Syt1 interaction.
It is remarkable that PERK cooperates with E-Syt1 to regulate lipid trafficking at EMCS. In addition to protein tethers maintaining close contact between the ER and mitochondria, ERshaping proteins are also required to sustain steady-state levels of PLs (Lahiri et al., 2014;Voss et al., 2012). Tricalbins (yeast orthologs of the mammalian E-Syt1/2/3) have been shown to utilize their curvature-generation and lipid-transport properties to facilitate the formation of cortical ER and ER-PM lipid transport (Collado et al., 2019). Hence, it is possible that membrane curvature and SMP properties of E-Syt1 enable PERK PLs trafficking at EMCS.
However, we cannot exclude that PERK and E-Syt1 interact indirectly and are part of a larger functional complex with other tethers either as separate entities or in partnership with this PERK complex. Consistent with this, severe PL transfer defects are observed in cells missing multiple components of the ERmitochondria contacts (Lahiri et al., 2014). An interesting partner could be Mitofusin-2 (MFN2), which is a known regulator of PERK (Muñoz et al., 2013) and was recently shown to favor PS transfer to mitochondria to foster PE synthesis (Hernández-Alvarez et al., 2019). These are interesting conjectures that need further exploration.
Recent studies reported that in response to acute ER stress or nutrient deprivation, PERK promotes protective mitochondrial remodeling and mitochondrial respiratory chain supercomplex formation through the coordination of transcriptional (ATF4-SCAF1 dependent) and translational signals (Balsa et al., 2019;Lebeau et al., 2018;Rainbolt et al., 2013). In response to cold stress or beta-adrenergic stimulation in brown adipose tissue, activated/phosphorylated PERK regulates thermogenesis by stimulating biogenesis of respiratory complexes and the MICOS multiprotein complex that controls mitochondria cristae junctions and bioenergetics (Kato et al., 2020;Latorre-Muro et al., 2021). While these studies reveal a role for PERK in key aspects of mitochondrial functions under ER stress conditions, it remained unclear whether PERK exhibited any noncanonical UPR function at EMCS. Although our study provides evidence that PERK regulates PL transfer at EMCS by scaffolding E-Syt1 through mechanisms that do not involve its role as UPR sensor, we cannot exclude that PERK has a complementary role in PL transfer by regulating proximity of these contact sites. The generation of PERK mutants abolishing its interaction with E-Syt1 while preserving PERK's role as regulator of the ER mitochondria contacts could help clarify this point.
However, our finding of a PERK-E-Syt1 axis functioning as a molecular effector of PL transfer between the ER and mitochondria further parses out how a pool of these ER proteins moonlight when present at EMCS to regulate mitochondrial lipid homeostasis and respiration. Indeed, supporting a role of PERK-E-Syt1 axis for PL trafficking and mitochondrial respiration, abolishing either the lipid transfer function of E-Syt1 or disrupting PERK-E-Syt1 interaction cause ER-mitochondria lipid transfer and mitochondrial respiratory capacity defects.
Finally, since mitochondrial and lipid metabolism aberrations are common denominators of devastating diseases like the Wolcott-Rallison syndrome caused by genetic mutations in PERK (EIF2AK3; Delépine et al., 2000), our study unravels a crucial new aspect in the growing repertoire of PERK functions.
To generate shPERK HeLa cells stably expressing the myc tagged kinase dead mutant (K622A), following viral particle production and transduction, HeLa cells that had efficiently integrated the pLenti K622A construct (K622A mutation in the µHsPERK-myc-pUC57 SC1010) were selected with hygromycin B (160 µg/ml).

Patient-derived cells
Skin-derived fibroblasts (Isabelle Meyts, Laboratory of Inborn Errors of Immunity, KU Leuven, Leuven, Belgium) were obtained from a healthy individual (CTR) and a patient with p.W681X/p.W681X nonsense mutation in EIF2AK3 (PERK) suffering from Wolcott-Rallison syndrome. Fibroblasts were isolated from a freshly obtained skin biopsy, treated overnight with collagenase Type III (Stemcell, 400 CDU/ml), plated in T25 culture flask in serum-free conditions. After the treatment, the cells were further maintained in Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 containing 4.5 g/l glucose, 0.11 g/l sodium pyruvate and 2 mM glutamine and supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin and 10% FBS.
All cells were maintained in 5% CO 2 at 37°C. Cells were routinely checked for mycoplasma contaminations using the Plasmotest kit (Invivogen) according to the manufacturer's instructions.

Western blotting
Samples were separated by SDS-PAGE on the Criterion system (Bio-Rad Laboratories) on a 4-12% Bis-TRIS gel and electrophoretically transferred to Protran 2 μm-pored nitrocellulose paper (PerkinElmer). The blots were blocked for 1 h at RT in TBS-T buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% Tween-20) containing 5% non-fat dry milk and then incubated with selected antibody solutions.
Samples were processed with appropriate secondary antibodies for chemical (HRP-based) detection (Cell Signaling Technologies) or Infrared detection (Thermo Fisher Scientific).
inhibitor [Pierce Protease Inhibitor Tablets, Thermo Fisher Scientific Inc.]) in MQ water. Cells were centrifuged to remove debris and 500 μg of proteins from the supernatant was combined with primary antibody/conjugated GFP antibody beads overnight (ON) at 4°C. Proteins were eluted with sample buffer (62.5 μM Tris-HCl, 10% glycerol, 2% SDS, 1x protease inhibitor, 1x phosphatase inhibitor (Pierce phosphatase Inhibitor Tablets, Thermo Fisher Scientific Inc.) in MQ water and loaded on gel for Western blot analysis. For developing IPs, ChemiDoc MP Imaging system and Rabbit DyLight 680/Mouse DyLight 800 secondary antibodies were used in cells transiently co-transfected with eGFP-empty vector or eGFP-tagged E-Syt1 and myc-tagged PERK-FL or myc-tagged PERK k618A ; for the rest of the experiments, HRP-based detection using as secondary antibody the Veriblot antibody was used in order to avoid the detection of the long and short chains of the IgG.
Aequorin measurements were acquired in KRB supplemented with 1 mM CaCl 2 , and the agonist was added to the same medium as indicated in figure legends. Cells were then lysed with Triton X-100 in a hypotonic Ca 2+ -rich solution (10 mM CaCl 2 in H2O), thus discharging the remaining aequorin pool. The light signal was collected and calibrated into [Ca 2+ ] values using the algorithm reported in Rizzuto et al. (1992).

HRP detection
HeLa cells expressing HRP-KDEL-myc were fixed on coverslips with 1.3% glutaraldehyde in 0.1 M cacodylate buffer, washed in 0.1 M ammonium phosphate [pH 7.4] buffer for 1 h, and HRP was visualized with 0.5 mg/ml DAB and 0.005% H2O2 in 0.1 M ammonium phosphate [pH 7.4] buffer. Development of HRP (DAB dark reaction product) took between 5 to 20 min and was stopped by extensive washes with cold water. Cells were postfixed in 2% OsO4+1% K3Fe(CN)6 in 0.1 M cacodylate buffer at 4°C for 1 h, washed in cold water and then contrasted in 0.5% uranyl acetate for 2 h at 4°C, dehydrated in an ethanol series and embedded in epon as for conventional EM. Ultrathin sections were counterstained with 2% uranyl acetate and observed under a FEI Tecnai 12 microscope, 4,800X magnification, equipped with a CCD (SiS 1kx1k keenView) camera and iTEM acquisition software.

Immunogold labeling
HeLa cells were fixed with a mixture of 2% PFA and 0.125% glutaraldehyde in 0.1 M phosphate buffer [pH 7.4] for 2 h and processed for ultracryomicrotomy as described previously (Slot and Geuze, 2007). Ultrathin cryosections were doubleimmunogold-labeled with antibodies (GFP, Life technologies, A11122, 1:100; PDI, Genetex, GTX30716, 1:500) and protein A coupled to 10 or 15 nm gold (CMC, UMC Utrecht, The Netherlands), as indicated in the legends to the figures. Immunogoldlabeled cryosections were observed under a FEI Tecnai 12 microscope, 6,800X magnification, equipped with a CCD (SiS 1kx1k keenView) camera and iTEM acquisition software.

FIB-SEM (focused on ion beam-scanning electron microscopy)
HeLa cells expressing HRP-KDEL-myc were grown on gridded coverslips and fixed in freshly prepared fixative (2% paraformaldehyde PFA, Applichem), 2.5% gluteraldehyde (GA, EMS) in 0.1 M sodium cacodylate (Sigma-Aldrich) buffer, pH 7.4) at RT for 30 min. Fixative was removed by washing 5 × 3 min in 0.1 M cacodylate buffer, and subsequently, samples were incubated in 20 mM glycine in 0.1 M sodium cacodylate for 5 min to quench unreacted glutaraldehyde. After another series of washes in 0.1 M sodium cacodylate, HRP in the samples was visualized with freshly prepared 0.5 mg/ml DAB and 0.005% H2O2 in 0.1 M sodium cacodylate. The reaction was followed under a light microscope and stopped when the staining was sufficiently clear by washing in cacodylate buffer. Next, samples were incubated in 1% osmium (OsO4, EMS), 1.5% potassium ferrocyanide (Sigma-Aldrich) in 0.1 M cacodylate buffer for 40 min at RT. After washing in ddH2O for 5 × 3 min, this was followed by overnight incubation at 4°C in 1:3 UAR in H2O (Uranyl Acetate Replacement [UAR], EMS). The next day, UAR was removed by washing in ddH2O for 5 × 3 min. After final washing steps, the samples were dehydrated using ice-cold solutions of increasing EtOH concentration (30, 50, 70, 90%, 2 × 100%), for 3 min each. Subsequent infiltration with resin (Spurr, EMS) was done by first incubating in 50% resin in ethanol for 2 h, followed by at least 3 changes of fresh 100% resin (including one overnight incubation). Next, samples were embedded in fresh resin and cured in the oven at 65°C for 72 h. For FIB-SEM imaging, embedded cells were mounted on aluminum SEM stubs (diameter 12 mm), and samples were coated with ∼20 nm of Platinum (Quorum Q150T ES). FIB-SEM imaging was performed using a Zeiss Crossbeam 540 system with Atlas5 software. The Focused Ion Beam (FIB) was set to remove 5-nm sections by propelling Gallium ions at the surface. Imaging was done at 1.5 kV using an ESB (back-scattered electron) detector.
Proximity Ligation Assay (PLA) PLA was performed using Duolink In Situ Red Started Kit Mouse/Rabbit (Duolink In Situ Detection Reagents Red, Sigma-Aldrich, DUO92008; Duolink In Situ PLA Probe Anti-Mouse MINUS, Sigma-Aldrich, DUO92004; Duolink In Situ PLA Probe Anti-Rabbit PLUS, Sigma-Aldrich, DUO92004; Duolink In Situ Mounting Medium with DAPI, Sigma-Aldrich, DUO82040) accordingly to the manufacturer's instructions. Briefly, after fixation with 4% PFA and permeabilization with 0.1% Triton, HeLa cells were incubated with primary antibody, VDAC1 (rabbit; ab15895) and IP3R3 (mouse; BD Bioscience 610312), in blocking solution (PBS + 0.1% Triton X-100 + 4% BSA) ON. Secondary antibodies PLUS and MINUS (anti-rabbit and anti-mouse IgG antibodies conjugated with oligonucleotides) were incubated 1:5 in blocking solution for 1 h at 37°C. The ligation solution (ligation buffer 1:5, ligase 1:40 in MQ) was then incubated for 30 min at 37°C. The amplification solution (amplification buffer 1:5, polymerase 1:40 in MQ) was incubated for 1 h 40 min at 37°C. After incubation, slides were mounted with Duolink In Situ Mounting Medium with DAPI. As a negative control, we incubated VDAC1 alone and with both PLUS and MINUS secondary antibodies.

Subcellular fractionation
Cells were collected and the resulting pellet after centrifugation (600-800 g for 5 min at RT) was resuspended in 5 ml of Starting Buffer 1 (SB1) containing 225 mM mannitol, 75 mM sucrose and 30 mM Tris-HCl, 0.1 mM EGTA, pH 7.4 and homogenized. Unbroken cells and nuclei were removed by centrifugation of the cell homogenate at 600 g for 5 min (at 4°C). The crude mitochondrial fraction (mito crude) was pelleted by centrifuging the supernatant at 7,000 g for 10 min at 4°C while the supernatant contained the ER. The crude mitochondria were resuspended in SB (225 mM mannitol, 75 mM sucrose and 30 mM Tris-HCl, pH 7.4) and after other two sequential centrifugations (7,000 g and 10,000 g for 10 min at 4°C), the obtained pellet was resuspended in 1 ml of mitochondria resuspending buffer MRB (250 mM mannitol, 5 mM HEPES and 0.5 mM EGTA), layered on top of a percoll gradient (30 and 15%) and spun down at 95,000 g for 40 min (using a Beckman ultracentrifuge, rotor SW41), to separate MAMs from the pure mitochondria (mito pure). Further ultracentrifugation (70Ti rotor, 100,000 g for 1 h at 4°C) was required to obtain the pellet of the MAMs fraction.
Mitochondrial mass (mitotracker green) Cells were washed, trypsinized, collected, and then stained with Mitotracker green 30 nM (M7514, Thermo Fisher Scientific) for 30 min. Samples were then washed two times with PBS and processed using BD FACSCanto II. Analysis was performed using FlowJo v10.8.1 software.

Mitochondrial bioenergetics
Cells were plated in a Seahorse XF24 (Agilent) Extracellular Flux Analyzer culture plate in culture medium overnight. Prior to the assay, cells were switched to the Mitostress assay medium (Seahorse Biosciences) supplemented with 25 mM galactose, 2 mM glutamine and 1 mM sodium pyruvate, following the manufacturer's instructions. OCR during the Mitostress assay were measured with the Seahorse XF24 (Agilent) Extracellular Flux Analyzer (Seahorse Biosciences) following the manufacturer's instructions. Following the Mitostress assay, 1 µM oligomycin, 0.5 µM FCCP (HeLa shCTR, shPERK, shPERK + PERK K618A , WT and DKO) or 2.5 µM FCCP (CTR and p. W681X) and 0.5 µM antimycin A diluted in assay medium were the final concentration in the wells. Once the run was finished, all the medium was removed from the well and all the cells were collected in 10 µl of Laemli buffer for BCA-based protein quantification.

NBD-PS live cells trafficking
After evaporating the chloroform from the solution NBD-PS (810194C, Avanti polar lipids) with N2 gas, NDB-PS was resuspended in ethanol to a concentration of 0.5 M. HeLa cells, were incubated with NBD-PS 1 mM for 30 min. After 30 min, NBD-PS was washed away and cells were incubated with Mi-toTracker Far Red (M22426 Thermo Fisher Scientific) for 30 min and then imaged 30 min later the NBD-PS staining. Human fibroblasts were incubated with NBD-PS 2.5 µM for 30 min in combination with MitoTracker Far Red and then imaged at t = 0. Images were recorded in live cells with Zeiss LSM 880 Airyscan (Pieter Vanden Berghe, University of Leuven, Bioimaging core VIB-KU Leuven, Sebastian Munck).
Image processing, analysis, and statistics For live imaging, confocal images colocalization analysis was performed using ImageJ/Fiji Software, JAcoP plugin, Manders coefficients. The mitochondrial morphology was analyzed with the macro "Mito-morphology" from ImageJ/Fiji Software.
PLA images were analyzed manually with "Threshold" and "Analyze particles" commands from ImageJ/Fiji Software (http://imagej.nih.gov/ij/). FIB-SEM images were first preprocessed by denoising using the BM4D algorithm (Maggioni et al., 2013). Then, images were first segmented manually with MIB (microscopy image browser), and then the manual segmentation was used to optimize a semi-automatic segmentation analysis. For the automated analysis, an iterative procedure based on U-Net (Falk et al., 2019) and manual correction was employed. First, annotations of the datasets were obtained manually for the mitochondria and semi-automatically for the ER (particularly thresholding, morphological cleaning, and manual correction). These annotations were used to train an initial 2D U-Net (3D volumes are processed slice by slice). Next, the dataset was segmented with this model, followed by small particle removal and manual correction. The U-Net model was then trained on the three annotated datasets to improve generalization performance and finally applied on the remaining datasets. These datasets were then post-processed by removing small particles and manual correction to obtain the final segmentation of the mitochondria and ER. Segmented images were then imported in Imaris 9.9 and analyzed with Surface-Surface contact plugin. In Imaris, Surface-area and volume were calculated using the Statistic features.
For the quantification of ER-mitochondria contact sites in HRP-stained Epon sections, the total circumference of each mitochondria and the length of the multiple HRP-positive ER segments closely associated (<30 nm) with them were measured by manual drawing using the iTEM software (Olympus), as in Galmes et al. (2016), on acquired micrographs of HeLa cells for each cell profile. Cells were randomly selected for analysis without prior knowledge of transfected plasmid or siRNA. All data are presented as mean (%) ± SEM.
For the quantification of E-Syt1 immunogold labeling on ultrathin cryosections, 300 gold particles were counted in randomly selected cell profiles and assigned to either reticular ER (ER), ER in contact with mitochondria (EMCS) and ER in contact with PM (ER-PM) using the iTEM software. All data are presented as mean (%) ± SEM of three technical replicates.
All data are represented as mean ± SEM. Data distribution was assumed to be normal but this was not formally tested. Statistical significance between two groups was determined by standard unpaired t-test with F-testing or one sample t test. Statistical significance between multiple groups was determined by one-way ANOVA to ensure comparable variance, then individual comparisons performed by Tukey's post-hoc test. Analysis was done in Prism v9.0f, GraphPad. *, P < 0.05; **, P < 0.01; ***, P <0.001; and ****, P <0.0001, where a P value <0.05 is considered significant.
Online supplemental material Fig. S1, related to Fig. 1 and Fig. 2, shows PERK levels in HeLa and fibroblast cell lines, mitochondrial morphology and mass analysis, immunoblots of cellular fractions, and lipidomics data. Fig. S2, related to Fig. 3, shows immunoblots of PSD, PSS1/ 2 enzymes, immunoblots of cellular fractions, protein quantifications, PLA assays, HRP-KDEL-myc EM analysis, and Ca 2+ measurements. Fig. S3, also related to Fig. 3, shows immunoblots of cellular fractions, lipidomics data, immunoblots of PSD, PSS1/2 enzymes, and mitochondrial mass analysis. Fig. S4, related to Fig. 4, shows immunoblots of cellular fractions, immunoblots of transiently transfected cell lines with GFP or GFPtagged E-Syt1 variants, protein quantifications, immunoblots of co-IP analysis, representative microscopy images and colocalization analysis between GFP-Esyt1/GFP-Esyt1ΔDE and STIM1-mCherry. Fig. S5, related to Fig. 5 and Fig. 6, shows representative microscopy images for the transfection efficiency of mCherry or mCherry-tagged E-Syt1 variants, immunoblots and E-Syt1 protein levels of electroporated cells with GFP or GFPtagged E-Syt1 variants. Video 1, Video 2, Video 3, and Video 4 show 3D videos of segmentations and reconstructions of FIB-SEM analysis. . The values plotted are the mean ± SEM from three biological replicates (n = 52 and n = 72 for shCTR and shPERK respectively) analyzed using unpaired Student's t test. Scale bar in overview image is 10 µm, and scale bar in magnification is 5 µm. (D) Mitotracker Green geometrical mean intensity (MFI) in shCTR and shPERK cells. The values plotted are the mean ± SEM from three biological replicates analyzed using unpaired Student's t test. (E) Representative immunoblot for PERK in CTR and p.W681X PERK mutant human fibroblasts. ACTIN serves as loading control. Arrows indicate truncated PERK and a nonspecific band. (F) Representative immunoblot for IP3R3, PERK, CNX, VDAC1, and CYTC from total lysates, mito crude, mito pure, and MAM fractions of shCTR and shPERK HeLa cells. (G) Representative immunoblot for PERK, CNX, VDAC1, and CYTC from total lysates, mito crude, mito pure, and MAM fractions of PERK +/+ and PERK −/− MEFs cells. Arrow indicates a non-specific band. (H) Abundance of PG from purified mitochondrial fractions of shCTR and shPERK HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from two biological replicates. (I) Abundance of DG from purified mitochondrial fractions of shCTR and shPERK HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from two biological replicates. (J) Abundance of PC, PI, PE, PG, PS from total cell lysates of shCTR and shPERK HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. NS = not significant. Source data are available for this figure: SourceData FS1. Figure S3. E-Syt1 is found at the EMCS and regulates mitochondrial phospholipid abundance. (A) Representative immunoblot for IP3R3, E-Syt1, CNX, VDAC1, and CYTC from total lysates, mito crude, mito pure, and MAM fractions of shCTR and shE-Syt1 HeLa cells. (B) Abundance of PG from purified mitochondrial fractions of shCTR and shE-Syt1 HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from two biological replicates. (C) Abundance of PG, PC, PI, PE, PS from total cell lysates of shCTR and shE-Syt1 HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (D) Abundance of total mitochondrial phospholipids from purified mitochondrial fractions of shCTR and shE-Syt1 cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from three biological replicates analyzed using one sample t test. (E) Abundance of DG from purified mitochondrial fractions of shCTR and shE-Syt1 HeLa cells, relative to control condition (shCTR). The values plotted are the mean ± SEM from two biological replicates. (F) Representative immunoblot for E-Syt1, PSD, PSS1, PSS2 in shCTR and shE-Syt1 HeLa cells and relative quantifications of PSD, PSS1, PSS2, and E-Syt1, normalized on ACTIN (loading control) and relative to control condition (shCTR). The values shown are the mean ± SEM from three biological replicates analyzed using one sample t test. (G) Mitotracker Green geometrical mean intensity (MFI) in shCTR and shE-Syt1 cells. The values plotted are the mean ± SEM from three biological replicates analyzed using unpaired Student's t test. NS = not significant. Source data are available for this figure: SourceData FS3.