A pair of transporters controls mitochondrial Zn2+ levels to maintain mitochondrial homeostasis

Ma, Tengfei; Zhao, Liyuan; Zhang, Jie; Tang, Ruofeng; Wang, Xin; Liu, Nan; Zhang, Qian; Wang, Fengyang; Li, Meijiao; Shan, Qian; Yang, Yang; Yin, Qiuyuan; Yang, Limei; Gan, Qiwen; Yang, Chonglin

doi:10.1007/s13238-021-00881-4

A pair of transporters controls mitochondrial Zn²⁺ levels to maintain mitochondrial homeostasis

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Published: 23 October 2021

Volume 13, pages 180–202, (2022)
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Protein & Cell

A pair of transporters controls mitochondrial Zn²⁺ levels to maintain mitochondrial homeostasis

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Tengfei Ma¹^na1,
Liyuan Zhao^1,2^na1,
Jie Zhang¹^na1,
Ruofeng Tang^1,2^na1,
Xin Wang¹,
Nan Liu¹,
Qian Zhang^1,2,
Fengyang Wang^1,2,
Meijiao Li¹,
Qian Shan¹,
Yang Yang¹,
Qiuyuan Yin¹,
Limei Yang¹,
Qiwen Gan^1,2 &
…
Chonglin Yang¹

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Abstract

Zn²⁺ is required for the activity of many mitochondrial proteins, which regulate mitochondrial dynamics, apoptosis and mitophagy. However, it is not understood how the proper mitochondrial Zn²⁺ level is achieved to maintain mitochondrial homeostasis. Using Caenorhabditis elegans, we reveal here that a pair of mitochondrion-localized transporters controls the mitochondrial level of Zn²⁺. We demonstrate that SLC-30A9/ZnT9 is a mitochondrial Zn²⁺ exporter. Loss of SLC-30A9 leads to mitochondrial Zn²⁺ accumulation, which damages mitochondria, impairs animal development and shortens the life span. We further identify SLC-25A25/SCaMC-2 as an important regulator of mitochondrial Zn²⁺ import. Loss of SLC-25A25 suppresses the abnormal mitochondrial Zn²⁺ accumulation and defective mitochondrial structure and functions caused by loss of SLC-30A9. Moreover, we reveal that the endoplasmic reticulum contains the Zn²⁺ pool from which mitochondrial Zn²⁺ is imported. These findings establish the molecular basis for controlling the correct mitochondrial Zn²⁺ levels for normal mitochondrial structure and functions.

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INTRODUCTION

Zinc, in the ion form (Zn²⁺), is an essential trace element for organisms. Zn²⁺ plays important roles in a wide range of physiological and cellular processes such as development, metabolism, DNA synthesis, and transcription (Colvin et al., 2010; Kambe et al., 2015). In humans, Zn²⁺ binds to about 10% of total proteins, acting either as a structural component of functional proteins (e.g. zinc fingers) or as a cofactor of catalytic enzymes including oxidoreductases, transferases, hydrolyases, lyases, isomerases, and ligases (Andreini et al., 2006; Colvin et al., 2010). In addition, Zn²⁺ functions as an important signaling ion (Frederickson et al., 2005; Yamasaki et al., 2007; Fukada et al., 2011). Intracellular Zn²⁺ promotes the expression and secretion of nerve growth factor (NGF) and early growth response factor 1 (EGR1), and promotes the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) (Park and Koh, 1999). In synapses, synaptic Zn²⁺ permeates Ca²⁺- and Zn²⁺-permeable GluR2-lacking AMPA receptors (AMPAR_Ca-Zns) and VGCCs (voltage-gated Ca²⁺ channels) to activate TrkB through either Src kinase- or metalloproteinase-dependent pathways, which in turn triggers synaptic potentiation (Huang et al., 2008). In C. elegans, physiological concentrations of Zn²⁺ were found to modulate Ras-mediated signaling (Bruinsma et al., 2002; Yoder et al., 2004). Zn²⁺ deficiency has been found to associate with poor growth and development, congenital neurological and immune disorders, defective wound healing, chronic inflammation, alopecia, persistent diarrhea, and bleeding (Kambe et al., 2015; Mammadova-Bach and Braun, 2019), to name a few. On the other hand, excess Zn²⁺ is highly toxic. Zn²⁺ accumulation is known to potently induce neuronal cell death in ischemia and blunt head trauma, and is associated with Alzheimer’s disease (AD) (Sensi et al., 2009).

Because either insufficient or excessive Zn²⁺ is deleterious to the cell, the cellular Zn²⁺ levels need to be tightly controlled. Cellular uptake and export of Zn²⁺ are carried out by two families of Zn²⁺ transporting proteins: Zrt, irt-related proteins (ZIPs), which are responsible for Zn²⁺ influx into the cytoplasm, and Zn²⁺ transporters (Znt), which mediate Zn²⁺ efflux from the cytosol (Kambe et al., 2015). These Zn²⁺ transporters usually localize to the plasma membrane or organelle membranes. In the cytosol, about 5%–15% of Zn²⁺ is sequestered by binding to metallothioneins (MTs), oxidation of which leads to release of free Zn²⁺ (Colvin et al., 2010; Kambe et al., 2015). In addition, cellular Zn²⁺ is compartmentalized in subcellular organelles, including the nucleus, endoplasmic reticulum (ER), Golgi, endosomes and lysosomes (Colvin et al., 2010). Shuttling of Zn²⁺ between these intracellular organelles and the cytosol is also mediated by Znt/ZIP transporters (Kambe et al., 2015). Surprisingly, it is not known to date how Zn²⁺ is transported into and out of mitochondria, the centers of cellular metabolism. Zn²⁺ is required for the activity of a wide variety of mitochondrial proteins, including OMA1 and YME1L, the enzymes responsible for cleavage of OPA1, which mediates the fusion of mitochondrial inner membranes (Head et al., 2009). It was found that ROS-induced lysosomal Zn²⁺ release and mitochondrial Zn²⁺ increase trigger mitochondrial division by promoting mitochondrial recruitment of Drp1 (Abuarab et al., 2017). A possible Drp1-ZIP1 interaction likely triggers the entry of Zn²⁺ into mitochondria, which reduces the mitochondrial membrane potential (MMP) and induces mitochondrial division or mitophagy (Cho et al., 2019). Mitochondria are known to play an important role in Zn²⁺ homeostasis. Mitochondrial uptake of Zn²⁺ promotes the clearance of cytosolic Zn²⁺ in neurons undergoing excitotoxicity (Paoletti et al., 1997; Dineley et al., 2005; Sensi et al., 2009). Nevertheless, abnormal accumulation of Zn²⁺ in mitochondria has multiple adverse effects by causing loss of mitochondrial membrane potential, inhibition of complexes I and III of the electron transport chain, inhibition of α-ketoglutarate dehydrogenase, and production of ROS (Dineley et al., 2003; Sensi et al., 2009). Zn²⁺ accumulation in mitochondria also activates mitochondrial permeability transition pore (MPTP), leading to release of proapoptotic factors and cell death (Jiang et al., 2001; Bossy-Wetzel et al., 2004; Malaiyandi et al., 2005a; Sensi et al., 2009). In addition, Zn²⁺ inhibits mitochondrial movement in neurons (Malaiyandi et al., 2005a). Elevated mitochondrial Zn²⁺ might also promote PINK/Parkin-mediated mitophagy in response to hypoxia-oxygenation (Bian et al., 2018).

Mitochondria are the powerhouses of the cell and coordinate the tricarboxylic acid (TCA) cycle, oxidative phosphorylation (OXPHOS), and ATP production to provide energy for cell survival and functions. At the same time, mitochondria are central to cell death, differentiation, and the innate immune response (Spinelli and Haigis, 2018). Mitochondria undergo many homeostatic processes, such as fusion, fission, mitophagy and the mitochondrial unfolded protein responses (UPR^mt), to maintain their structure and functions (Youle and van der Bliek, 2012; Mishra and Chan, 2016; Pickles et al., 2018). Despite the fact that many mitochondrial proteins require the presence of Zn²⁺ for their functions, it is largely unknown how mitochondrial structure and functions are affected when mitochondrial Zn²⁺ homeostasis is disrupted. Particularly, the identities of mitochondrion-localized Zn²⁺ transporters remain to be revealed. In this study, we use C. elegans as a model to demonstrate that SLC-30A9/Znt-9 is the mitochondrial Zn²⁺ exporter, loss of which causes mitochondrial Zn²⁺ accumulation and disruption of mitochondrial structure and functions. We further identify the Ca²⁺-activated Mg²⁺-ATP transporter SLC-25A25 as an important regulator of mitochondrial Zn²⁺ import, loss of which suppresses the accumulation of mitochondrial Zn²⁺. Moreover, we reveal that the ER serves as the Zn²⁺ reservoir, from which mitochondrial Zn²⁺ is imported. Our findings thus provide molecular insights into the mechanisms that control mitochondrial Zn²⁺ levels for maintenance of mitochondrial homeostasis.

RESULTS

Loss of slc-30A9 leads to mitochondrial abnormality

To identify regulators that are important for mitochondrial homeostasis, we performed genetic screens to search for mutants that display abnormal mitochondrial morphology, using C. elegans adult animals carrying an integrated array (yqIs157) that expresses mitochondrion-targeted GFP (Mito-GFP) in hypodermal cells (Zhou et al., 2019; Tang et al., 2020). Among the mutants we obtained, 5 of them (yq158, yq166, yq172, yq189, yq212) displayed similar abnormally enlarged spherical Mito-GFP-positive structures with varying sizes in the hypodermis, in contrast to the predominantly tubular Mito-GFP-labeled structures in wild-type (N2) animals (Fig. 1A). These mutants failed to complement one another, suggesting that they affected the same gene. We thus used the yq158 mutant to carry out further analysis. In yq158 animals, the abnormal spherical structures were also positive for mCherry-tagged TOMM-20 (TOMM-20::mCh, outer mitochondrial membrane) (Fig. 1B) and GFP-fused F54A3.5 (F54A3.5::GFP, the Mic10 subunit of the MICOS complex, inner mitochondrial membrane) (Fig. S1A). This indicates that the large spherical structures were indeed abnormal mitochondria. Using another integrated array, hqIs181, which expresses GFP fused with the mitochondrial localization sequence (mtLS::GFP) in multiple tissues (Zhou et al., 2019), we found that abnormal spherical mitochondria also existed in muscle and intestinal cells in yq158 mutants, in contrast to the tubular mitochondria in these tissues in N2 animals (Fig. 1C). To determine how mitochondrial ultrastructure was affected, we performed transmission electron microscopy (TEM) analysis. N2 animals contained tubular or small globular mitochondria in both hypodermal and muscle cells. In contrast, yq158 mutants had greatly enlarged mitochondria with severely damaged cristae (Fig. 1D), consistent with the observations using fluorescence markers. Under TEM, abnormal mitochondria were observed in the germline, sperm, oocytes and embryos in yq158 mutants (Fig. S1B). In addition, we examined the time-course of appearance of abnormal mitochondria in larval development. In N2 animals, mitochondria in the hypodermis were mostly tubular and well connected from larval stage 1 (L1) to adults. In yq158 mutants, however, the abnormal spherical mitochondria were observed as early as in L1 animals, and became predominant in adults (Fig. S1C). RNA interference (RNAi) of eat-3/OPA1 (Kanazawa et al., 2008), the gene required for fusion of mitochondrial inner membranes, essentially abolished mitochondrial enlargement in yq158 mutants, while double mutants of yq158 with tm1108, a deletion mutant of the drp-1 gene required for mitochondrial fission (Labrousse et al., 1999), exhibited abnormally enlarged mitochondria (Fig. S1D). Thus, mitochondrial fusion is required for the abnormal enlargement of mitochondria in yq158 mutants. Taken together, these findings suggest that the gene affected in yq158 and the other 4 mutants is important for mitochondrial homeostasis.

We mapped yq158 and the other 4 mutations to linkage group III (LGIII) and identified the affected gene, Y71H2AM.9, which encodes a protein that is homologous to the mammalian putative Zn²⁺ transporter SLC30A9 and the E. coli Zn²⁺ transporter Yiip (Figs. 1E and S2) (Lu and Fu, 2007). We thus renamed the Y71H2AM.9 gene slc-30A9. While yq166, yq172 and yq212 caused changes of amino acid residues, the yq158 mutation caused a premature translation termination at Trp305 (W305stop) in the encoded protein. In addition, the yq189 mutation affected a splicing site in slc-30A9 mRNA (Fig. 1E). Transgenic expression driven by the slc-30A9 promoter of wild-type slc-30A9, but not mutant slc-30A9 containing the yq158 mutation (W305stop), fully rescued the abnormal spherical mitochondria in slc-30A9 (yq158) mutants to the wild-type level in multiple tissues (Fig. 1F). Similar expression of GFP-fused SLC-30A9 (SLC-30A9::GFP), which is widely expressed in multiple tissues (Fig. S1E), also rescued the enlargement of mitochondria labeled with TOMM-20::mCh (Fig. 1G). SLC-30A9::GFP colocalized with TOMM-20::mCh (Fig. 1G). When expressed in HeLa cells, GFP-fused C. elegans SLC-30A9 and GFP-fused human SLC30A9 were both found to localize to mitochondria stained with MitoTracker DR (Fig. S3A and S3C). Taken together, these findings suggest that SLC-30A9 localizes to mitochondria and plays an essential role in maintaining mitochondrial structure and functions.

SLC-30A9 is a mitochondrial Zn²⁺ exporter

By prediction, SLC-30A9 has a similar structure to that of the bacterial Yiip Zn²⁺ transporter (Lu and Fu, 2007). We thus examined whether SLC-30A9 has Zn²⁺ binding activity. In microscale thermophoresis (MST) assays, SLC-30A9-EGFP expressed in HEK293 cells bound with Zn²⁺, but not Ca²⁺, Mg²⁺ (Figs. 2B, S3A and S3B). SLC-30A9-EGFP probably bound with Cu²⁺ and Mn²⁺ with low affinity (Fig. S3A and S3B). The Zn²⁺ binding activity localizes to the C-terminal part containing the cation-efflux domain, while the N-terminus has no obvious Zn²⁺ binding activity (Fig. 2B). Mitochondrion-localized human SLC30A9-GFP similarly bound to Zn²⁺ (Fig. S3C and S3D). We next mutated SLC-30A9 residues corresponding to those amino acids important for Zn²⁺ transporter activity in bacterial Yiip, including H198, D202, D221, and H402 (Figs. 2A and S2) (Lu and Fu, 2007), and examined how this affected the rescuing effect on slc-30A9 (yq158) mitochondria. When expressed under the control of the hypodermis-specific col-19 promoter, wild-type SLC-30A9 fully rescued the mitochondrial defects in slc-30A9 (yq158) hypodermis (Fig. 2C). However, similarly expressed SLC-30A9 (H198A/D202A) or SLC-30A9 (H402A) mutants had strongly reduced rescuing activity, and SLC-30A9 (D221A) had no rescuing activity (Fig. 2C). These results suggest that the Zn²⁺ transporter activity of SLC-30A9 is required for normal mitochondrial structure.

We next examined mitochondrial Zn²⁺ levels in slc-30A9 mutants. We purified mitochondria from both N2 and slc-30A9 (yq158) mutants (Fig. 2D and 2E) and assessed Zn²⁺ levels using ICP-MS (inductively coupled plasma mass spectrometry). In slc-30A9 (yq158) mutants, the mitochondrial Zn²⁺ level was >7 times higher than in N2 animals (Fig. 2F). Other divalent cations, including Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Co²⁺, and Cd²⁺, were not significantly changed (Fig. 2F). Thus, loss of slc-30A9 caused mitochondrial Zn²⁺ accumulation. To corroborate this result, we expressed a mitochondrion-localized Zn²⁺ sensor, Mito-eCALWY-4 (Chabosseau et al., 2014), to detect mitochondrial Zn²⁺ in C. elegans using FRET and FLIM-FRET assays (Gordon et al., 1998; Sun et al., 2011; Lin et al., 2015). In the absence of Zn²⁺, the eCALWY-4 sensor produces fluorescence resonance energy transfer, with a stronger normalized measure of FRET (FRETN) but a shorter florescence lifetime (τ_c) of the donor fluorescent protein (CFP). When bound to Zn²⁺, eCALWY-4 generates a weaker FRETN while the fluorescence lifetime (τ_c) of the donor is increased (Fig. 2G) (Gordon et al., 1998; Chabosseau et al., 2014; Lin et al., 2015). In the FRET and FLIM-FRET assays, slc-30A9 (yq158) mutants had much weaker FRETN (measured by FRET) but significantly longer τ_c (measured by FLIM-FRET) of Mito-eCALWY-4 than N2 animals (Fig. 2H). This suggests that Zn²⁺ accumulated in and likely caused abnormality of slc-30A9 (yq158) mitochondria. Supporting this notion, the abnormal mitochondrial enlargement in slc-30A9 (yq158) mutants was abrogated when the worms were treated with the Zn²⁺ chelator, TPEN (N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine) (Fig. 2I). Collectively, the above results revealed that SLC-30A9 is a mitochondrial Zn²⁺ exporter required for mitochondrial Zn²⁺ efflux.

Loss of slc-30A9 impairs mitochondrial function, compromises animal development and shortens the life span

We next assessed how mitochondrial Zn²⁺ accumulation affects mitochondrial function and animal development. slc-30A9 mutants had greatly reduced mitochondrial membrane potentials, and their ATP levels were strongly decreased (Fig. 3A and 3B). Consistent with the reduction of ATP levels, the animal bending frequency of slc-30A9 mutants was significantly lower than that of N2 animals (Fig. 3C). Remarkably, slc-30A9 mutants displayed strong mitochondrial unfolded protein responses (UPR^mt), as evidenced by the strongly increased signals of GFP driven by the hsp-6 promoter (P_hsp-6GFP) in slc-30A9 mutants (Nargund et al., 2015; Lin et al., 2016) (Fig. 3D). In addition, slc-30A9 mutants had far fewer progeny, indicating that loss of slc-30A9 impairs fertility (Fig. 3E). Around 30% of slc-30A9 mutant embryos died (Fig. 3F), and the development time from embryo to larval stage 4 (L4) of slc-30A9 mutants was nearly 2 times that of N2 animals (Fig. 3G). Moreover, the average life span of slc-30A9 adults was decreased by >50% compared to N2 animals (Fig. 3H). Taken together, these data suggest that, in the absence of slc-30A9, mitochondrial Zn²⁺ accumulation results in mitochondrial dysfunction and pleotropic defects of C. elegans development and physiology.

Mutations in slc-25A25 suppress the abnormal mitochondrial enlargement in slc-30A9 mutants

To investigate further how mitochondrial Zn²⁺ homeostasis is maintained, we carried out slc-30A9 suppressor screens using two strategies. First, we mutagenized slc-30A9 (yq158) animals and searched for mutants that displayed tubular mitochondria in adult hypodermis. Two mutants, yq350 and yq351, were obtained. In double mutants of yq350 or yq351 with slc-30A9 (yq158), mitochondria exhibited predominantly tubular morphology in multiple tissues rather than the abnormally enlarged spherical mitochondria in slc-30A9 (yq158) single mutants (Fig. 4A). Second, we performed a suppressor screen on slc-30A9 (yq158) expressing P_hsp-6GFP to look for mutants with greatly reduced UPR^mt signals. Three mutants, yq371, yq372, and yq373, had strongly reduced GFP (UPR^mt) signals when doubled with slc-30A9 (yq158) (Fig. 4B). Using SNP mapping and sequencing, we unexpectedly found that all 5 of these mutants affected the same gene, F17E5.2, on linkage group X (LGX). Within the F17E5.2 gene, yq371 caused a mis-splicing mutation, while the other 4 mutations changed amino acid residues in the encoded protein (Fig. 4C). In addition, we used the CRISPR/Cas9 system to generate another mutant, yq406, which carried an early stop-codon mutation in F17E5.2 (Fig. 4C). Double mutants of yq406 with slc-30A9 (yq158) had tubular mitochondria and strongly reduced UPR^mt, confirming that loss of F17E5.2 suppressed the abnormal enlargement of mitochondria in the absence of slc-30A9 (Fig. 4A and 4B).

F17E5.2 encodes a homolog of mammalian SLC25A25/SCaMC-2 (short calcium-dependent mitochondrial carrier 2), a Ca²⁺-activated mitochondrial inner membrane Mg²⁺-ATP transporter with 4 EF-hands (EFhs) and 3 mitochondrial carrier domains (Figs. 4C and S4A) (Joyal and Aprille, 1992; del Arco and Satrustegui, 2004; Fiermonte et al., 2004; Yang et al., 2014; Hofherr et al., 2018). By predication, F17E5.2 only has 3 EFhs (Figs. 4 C and S4A). F17E5.2 was thus renamed slc-25A25. Transgenic expression of slc-25A25 under the control of its own promoter or the hypodermal Y37A1B.5 promoter restored the abnormal mitochondrial enlargement as well as the strong UPR^mt in slc-30A9;slc-25A25 double mutants (Fig. 4D and 4E). These findings provide further evidence that slc-25A25 is responsible for rescue of slc-30A9-induced mitochondrial defects. Like SLC-30A9, SLC-25A25 expression was seen in multiple tissues (Fig. S4B). In addition, mitochondrion-localized SLC-25A25::GFP was observed in N2 animals and was more evident in slc-30A9(yq158);slc-25A25 (yq406) double mutants (Fig. 4D), which suggests that SLC-25A25 likely functions on mitochondria.

SLC-25A25 is important for normal mitochondrial structure and function

We next investigated whether slc-25A25 mutations caused mitochondrial defects. Although loss of slc-25A25 suppressed the abnormal enlargement of spherical mitochondria in slc-30A9 animals, the mitochondria in slc-25A25 (yq406) single mutants appeared as abnormally flattened tubules in multiple tissues compared with N2 animals (Fig. 5A). slc-25A25 (yq406) mutants treated with RNAi of drp-1 or eat-3 displayed similar mitochondrial defects to N2 animals with the same RNAi treatment (Fig. S4C), suggesting that mitochondrial fusion and fission are important for the mitochondrial defects caused by loss of slc-25A25. Using TEM, we found that mitochondria in slc-25A25 (yq406) hypodermal cells were morphologically abnormal compared with those in N2 animals (Fig. 5B). In slc-25A25 (yq406) muscle cells, mitochondria contained vacuolar structures (Fig. 5B). The slc-25A25 mutants did not have an obvious UPR^mt (Fig. 5C), consistent with the finding that loss of slc-25A25 suppressed the UPR^mt induced by slc-30A9 loss of function (Fig. 4B).

We further analyzed the effect of slc-25A25 mutations on mitochondrial functions and animal development. The slc-25A25 mutants yq406 and yq351 exhibited significantly reduced mitochondrial membrane potential and ATP levels, and the animal bending rates were significantly decreased (Fig. 5D–F). In addition, slc-25A25 mutants generated fewer progeny, displayed about 10% embryonic lethality, and had slower development (Fig. 5G–I). Nevertheless, slc-25A25 adults appeared to have a similar life span to N2 animals (Fig. 5J). Importantly, compared with slc-30A9 (yq158) single mutants, double mutants of slc-25A25 (yq406) with slc-30A9 (yq158) exhibited nearly normal mitochondrial structures (Fig. 5B), enhanced ATP production and motor ability (Fig. 5E and 5F), more progeny, decreased embryonic lethality, and faster development (Fig. 5G–I). The median life span of the double mutants was significantly enhanced compared with slc-30A9 (yq158) single mutants (Fig. 5J). Taken together, these results suggest that SLC-25A25 is important for normal mitochondrial structure and functions, and that slc-25A25 functions to antagonize slc-30A9.

SLC-25A25 binds with Zn²⁺ and is important for mitochondrial Zn²⁺ accumulation in slc-30A9 mutants

Using the Mito-eCALWY-4 Zn²⁺ sensor and FLIM-FRET, we assessed whether loss of slc-25A25 suppressed the mitochondrial Zn²⁺ accumulation caused by loss of slc-30A9. Compared with N2 animals, the donor fluorescence lifetime (τ_c) of Mito-eCALWY-4 in slc-30A9 (yq158) animals was strongly increased (Figs. 2H and 6A), indicating the elevated mitochondrial Zn²⁺ level. In slc-30A9 (yq158);slc-25A25 (yq406) double mutants, however, the donor fluorescence lifetime (τ_c) in Mito-eCALWY-4 was significantly reduced (Fig. 6A). Thus, loss of slc-25A25 inhibited the mitochondrial Zn²⁺ accumulation in slc-30A9 mutants, which suggests that SLC-25A25 regulates mitochondrial Zn²⁺ levels. To investigate this further, we examined whether SLC-25A25 binds with Zn²⁺. In MST assays, purified recombinant worm SLC-25A25-EGFP bound with Zn²⁺ (K_d 2.40 ± 0.27 μmol/L) (Fig. 6B). Likewise, recombinant human SLC25A25-EGFP bound with Zn²⁺ (K_d 1.87 ± 0.31 μmol/L). EGFP-tagged C. elegans SLC-25A25 and human SLC25A25 both bound Ca²⁺ with similar affinity (Fig. 6B and 6C). As a negative control, recombinant EGFP had no obvious Zn²⁺- or Ca²⁺- binding activity (Fig. 6D). These findings suggest that both C. elegans SLC-25A25 and human SLC25A25 can bind with Zn²⁺ in addition to Ca²⁺. Notably, transgenic expression of GFP-fused human SLC25A25 driven by the C. elegans slc-25A25 promoter restored the mitochondrial enlargement in slc-30A9 (yq158);slc-25A25 (yq406) double mutants (Fig. 6E), suggesting an evolutionarily conserved function of SLC25A25. Interestingly, double mutants of slc-30A9 (yq158) with mcu-1 (ju1154) (Xu and Chisholm, 2014), a strong loss-of-function mutant of the worm mitochondrial calcium uniporter (MCU), exhibited a similar mitochondrial abnormality to slc-30A9 (yq158) single mutants (Fig. 6F). This suggests that MCU is not responsible for the mitochondrial enlargement induced by loss of slc-30A9.

SLC-25A25/SLC25A25 overexpression leads to mitochondrial enlargement and Zn²⁺ elevation

To investigate whether C. elegans SLC-25A25 and human SLC25A25 promote mitochondrial Zn²⁺ import, we generated HeLa cell lines expressing EGFP-tagged SLC-25A25 (worm) or SLC25A25 (human). We then purified mitochondria and measured divalent ions using ICP-MS. Our results indicated that mitochondrial Zn²⁺ levels were strongly increased in cells expressing EGFP-SLC-25A25 (worm) or EGFP-SLC25A25 (human) (Fig. 7A and 7B). No obvious changes were detected for other divalent ions including Cu²⁺, Fe²⁺, Mg²⁺, Mn²⁺, Co²⁺ and Ca²⁺ (Fig. 7A and 7B). Compared with the control cells, the cells expressing worm SLC-25A25 or human SLC25A25 had significantly enlarged mitochondria, which were further enlarged by supplementation of the culture medium with 0.1 mmol/L Zn²⁺ (Fig. 7C). Furthermore, the enlarged mitochondria were strongly positive for the Zn²⁺ indicator Zinpyr-1, which indicates that they contained elevated levels of Zn²⁺ compared with the mitochondria in control cells (Fig. 7D). Collectively, these results provided further evidence that SLC-25A25 promotes mitochondrial import of Zn²⁺.

Inhibiting the ER-localized Zn²⁺ transporter SLC-30A5 suppresses the mitochondrial Zn²⁺ accumulation in slc-30A9 mutants

Because cellular Zn²⁺ is compartmentalized (Colvin et al., 2010), we sought to determine the location of the Zn²⁺ pool that provides the Zn²⁺ for mitochondria. To do this, we performed a candidate RNAi screen to knock down putative C. elegans Zn²⁺ transporters (Table S2). RNAi of slc-30A5/ZnT5 strongly inhibited the abnormal enlargement of spherical mitochondria in slc-30A9 (yq158) mutants (Fig. 8A). TEM analysis revealed that slc-30A5 RNAi-treated slc-30A9 (yq158) animals exhibited tubular mitochondria in hypodermal cells, in contrast to the abnormally enlarged mitochondria in control RNAi-treated slc-30A9 (yq158) mutants (Fig. 8B). SLC-30A5/Znt-5 is predicted to be a Zn²⁺ transporter localized to the endoplasmic reticulum (ER). In line with this, EGFP-fused SLC-30A5 (EGFP-SLC-30A5) ectopically expressed in HeLa cells co-localized with the ER marker KDEL-mCh, and bound with Zn²⁺ in MST assays (Fig. 8C). In C. elegans, transgenic SLC-30A5::GFP under the control of the slc-30A5 promoter was found to co-localize with the ER marker SPCS-1 tagged with mCherry (SPCS-1::mCh) (Fig. 8D). In FLIM-FRET assays, slc-30A5 RNAi treatment significantly reduced Zn²⁺ levels in both ER and mitochondria in N2 and slc-30A9 (yq158) animals, as evidenced by the reduction in the fluorescence lifetime of CFP (τ_c) of ER- and Mito-eCALWY-4 probes (Fig. 8E). These findings suggest that mitochondrial Zn²⁺ is imported from the ER. Using the ER-eCALWY-4 Zn²⁺ sensor, we found that slc-25A25 (yq406) animals as well as slc-30A9 (yq158);slc-25A25 (yq406) double mutants had significantly higher ER Zn²⁺ levels (higher τ_c from the ER-eCALWY probe) than the wild type (Fig. 8F). Furthermore, in both slc-25A25 (yq406) single mutants and slc-30A9 (yq158);slc-25A25 (yq406) double mutants, slc-30A5 RNAi significantly reduced the Zn²⁺ levels in both the ER and mitochondria, as evidenced by the decreased τ_c from the ER- and Mito-eCALWY-4 probes (Fig. 8G). Thus, decreasing SLC-30A5-mediated Zn²⁺ uptake into the ER reduced the mitochondrial Zn²⁺ levels, suggesting that the ER likely serves as the Zn²⁺ pool for mitochondrial Zn²⁺ import.

We investigated the physiological requirement for the ER Zn²⁺ pool in mitochondrial functions. RNAi of slc-30A5 restored the mitochondrial structure in slc-39A9 (yq158) mutants (Fig. 8B). Furthermore, mitochondrial membrane potentials were greatly enhanced by slc-30A5 RNAi in yq158 and other mutants of slc-30A9, as indicated by Mito-CMXRos staining (Fig. 8H). In slc-30A9 mutant animals, the ATP levels and the body bending frequencies were also significantly improved by slc-30A5 RNAi (Fig. 8I and 8J). In addition, slc-30A5 RNAi significantly reduced the embryonic lethality and shortened the larval developmental time of slc-30A9 mutants (Fig. 8K and 8L). These results suggest that reducing SLC-30A5-mediated ER Zn²⁺ import ameliorated the structural and functional mitochondrial defects in slc-30A9 mutants.

ZnT5 inactivation ameliorates SLC-25A25-related mitochondrial defects

To consolidate the findings that the ER provides the Zn²⁺ pool for mitochondrial Zn²⁺, we treated HeLa cells expressing worm SLC-25A25 or human SLC25A25 with siRNA (small RNA interference) of ZnT5, the human ortholog of C. elegans SLC-30A5. While ZnT5 siRNA did not obviously change mitochondrial morphology in control cells, the Zn²⁺-induced mitochondrial enlargement in cells expressing GFP-SLC-25A25 (worm) or GFP-SLC25A25 (human) was strongly inhibited (Fig. 9A and 9B). Moreover, ZnT5 siRNA significantly reduced the elevation in mitochondrial Zn²⁺ levels, as evidenced by the reduced mitochondrial fluorescence intensity of the Zn²⁺ reporter Zinpyr-1 (Fig. 9C). These results indicated that SLC-25A25/SLC25A25-mediated mitochondrial Zn²⁺ import depends on ZnT5-mediated ER Zn²⁺ uptake, thus providing further evidence that the ER serves as the Zn²⁺ pool for mitochondrial Zn²⁺ import. Supporting this conclusion, SLC-25A25::GFP (worm) and EGFP-SLC25A25 (human) were found at mitochondrion-ER contacts in worm and HeLa cells, respectively (Fig. 9D and 9E). In slc-30A9 (yq158);slc-25A25 (yq406) double mutants, the functional SLC-25A25::GFP was even more obviously enriched at mitochondrion-ER contacts (Fig. 9D). Taken together, these findings suggest that the ER contains the Zn²⁺ pool for mitochondrial Zn²⁺ import, and that mitochondrial import of Zn²⁺ from the ER is probably achieved through mitochondrion-ER contacts.

DISCUSSION

Although Zn²⁺ is known to play a critical role in mitochondrial homeostasis, it was not understood previously how mitochondrial Zn²⁺ is controlled at an appropriate level. In this study, we used unbiased genetic screens, combined with targeted gene screening, to reveal the identities of mitochondrial transporters that control mitochondrial Zn²⁺ homeostasis (Fig. 9F). Our findings demonstrated that SLC-30A9 is the mitochondrial Zn²⁺ exporter. This is evidenced by the facts that SLC-30A9 localizes to mitochondria and binds with Zn²⁺, and its functional loss causes a strong elevation in mitochondrial Zn²⁺ levels and consequently severe damage of mitochondrial structure and functions. Loss of slc-30A9 strongly impaired animal development, as revealed by the findings that slc-30A9 mutants exhibit severely reduced fertility, embryonic lethality and retardation of larval development. In addition, Zn²⁺-induced mitochondrial damage greatly reduced the life span of slc-30A9 mutants. These results suggest that SLC-30A9-mediated mitochondrial export of Zn²⁺ is critical for mitochondrial homeostasis, animal development and life span.

Mitochondrial calcium uniporter (MCU) was suggested to mediate mitochondrial entry of Zn²⁺ (Malaiyandi et al., 2005b; Medvedeva and Weiss, 2014; Cho et al., 2019). However, genetic inactivation of mcu-1, which encodes the C. elegans homolog of mammalian MCU, did not suppress the abnormal mitochondrial enlargement in slc-30A9 mutants. This suggests that the mechanism of mitochondrial Zn²⁺ import is independent of MCU. Using two suppressor screen strategies, we revealed that mutations in slc-25A25 suppressed the severe mitochondrial defects of slc-30A9 mutants. Our results further indicated that both C. elegans and human SLC25A25 localize to mitochondria and bind with Zn²⁺. Moreover, SLC25A25 functions in an evolutionarily conserved manner, as heterologous expression of human SLC25A25 successfully substituted the function of C. elegans SLC-25A25 in double mutants of slc-25A25 with slc-30A9. Loss of slc-25A25 significantly reduced mitochondrial accumulation of Zn²⁺ in slc-30A9 mutants. In addition, both C. elegans and human SLC25A25 bind to Ca²⁺. These findings suggest that SLC-25A25 is a Ca²⁺-binding regulator of mitochondrial Zn²⁺ import. Importantly, ectopic expression of C. elegans SLC-25A25 or human SLC25A25 caused enlargement of mitochondria with elevation of mitochondrial Zn²⁺ levels. This provides strong evidence that SLC25A25 promotes mitochondrial Zn²⁺ import, in addition to its originally reported function as a mitochondrial inner membrane Mg²⁺-ATP/Pi transporter (del Arco and Satrustegui, 2004; Fiermonte et al., 2004; Satrustegui et al., 2007) . Altogether, these findings suggest that SLC-25A25 and SLC-30A9 are a pair of mitochondrial transporters which control mitochondrial Zn²⁺ import and export, respectively, to maintain the mitochondrial Zn²⁺ levels required for normal mitochondrial structure and functions (Fig. 9F). It was reported that human and plant ATP-Mg/phosphate carriers (APCs), such as hAPC1/SLC25A24 and Arabidopsis APC3, co-transport divalent cations including Mg²⁺, Mn²⁺, Fe²⁺, Zn²⁺, and Cu²⁺ in proteoliposome assays (Monne et al., 2017). In our study, ectopic expressed human and C. elegans SLC25A25 greatly increased mitochondrial Zn²⁺ levels, suggesting that SLC25A25 is very likely to transport Zn²⁺, in addition to its function as an ATP-Mg transporter. Further studies will be needed to demonstrate whether SLC25A25 functions as a Zn²⁺ transporter or an ATP-Mg/phosphate transporter that also transports zinc. It also remains to be determined whether Zn²⁺ is transported in complex with ATP.

Using targeted gene screening, we found that inactivation of slc-30A5/ZnT5 strongly suppressed the enlargement of mitochondria in slc-30A9 mutants. In both C. elegans and HeLa cells, worm SLC-30A5 and human SLC30A5/ZnT5 localize to the ER and bind Zn²⁺, suggesting that they function on the ER for Zn²⁺ transport. We found that RNAi of slc-30A5/ZnT5 significantly reduced the Zn²⁺ levels in both ER and mitochondria in C. elegans. This suggests that SLC-30A5 functions as an ER Zn²⁺ importer and that the ER serves as the Zn²⁺ reservoir from which mitochondrial Zn²⁺ is imported. Supporting this conclusion, we found that siRNA knockdown of ZnT5 significantly inhibited the abnormal mitochondrial enlargement and elevated mitochondrial Zn²⁺ levels in HeLa cells ectopically expressing C. elegans SLC-25A25 or human SLC25A25. Our findings suggest that C. elegans SLC-25A25 plays an important role in mitochondrial Zn²⁺ import, consistent with other studies that mammalian SLC25A25 localizes to the inner mitochondrial membrane (Tewari et al., 2012). We reason that the transfer of Zn²⁺ from ER to mitochondria is achieved through ER-mitochondrion contacts (Fig. 9F). It is possible that another ER transporter is responsible for exporting Zn²⁺ at the contacts, and the Zn²⁺, either alone or in complex with ATP, is then swiftly imported into mitochondria by SLC-25A25. Supporting this hypothesis, SLC-25A25 was found to be enriched at ER-mitochondrial contacts when slc-30A9 is deficient (Fig. 9D). Nevertheless, our unbiased genetic screen and targeted gene screen did not identify any ZnT-type Zn²⁺ exporters on the ER. In Drosophila, SLC25A25 was found to act downstream of the ER-localized Ca²⁺-permeable channel TRPP2 (transient receptor potential channel polycistin-2) to mediate TRPP2 signaling to regulate mitochondrial metabolism (Hofherr et al., 2018). In the case of Zn²⁺ transfer from the ER to mitochondria, further investigation is needed to determine whether TRPP2 or other transporters act upstream of SLC-25A25 to export Zn²⁺ out of the ER.

A single mutation in SLC30A9 was recently found to be associated with an autosomal recessive cerebro-renal syndrome, which manifests as neurological deterioration, intellectual disability, ataxia, camptocormia, oculomotor apraxia, and nephropathy (Perez et al., 2017). However, the molecular and cellular basis for this genetic disorder remains unknown. Deregulation of SLC30A9 expression was also reported for hepatocellular carcinoma (HCC) (Gartmann et al., 2018) and prostate cancer (PCa) (Singh et al., 2016). Likewise, SLC25A25 expression was found to be mis-regulated in fulminant type I diabetes (Ye et al., 2020), HCCs (Kido and Lau, 2019) and hypospadias (Karabulut et al., 2013). Our findings that SLC-30A9 and SLC-25A25 cooperate to control mitochondrial Zn²⁺ levels thus provide important mechanistic insights for understanding these human diseases.

MATERIALS AND METHODS

C. elegans strains and genetics

C. elegans strains were grown and maintained on NGM medium seeded with E. coli OP50 at 20 °C. The Bristol strain N2 was used as the wild type. The Hawaiian strain CB4856 was used in single nucleotide polymorphism (SNP) mapping. slc-30A9 (yq158), slc-30A9 (yq166), slc-30A9 (yq172), slc-30A9 (yq189), slc-30A9 (yq212), slc-30A9 (yq158);slc-25A25 (yq350), slc-30A9 (yq158);slc-25A25 (yq351), slc-30A9 (yq158);slc-25A25 (yq371), slc-30A9 (yq158);slc-25A25 (yq372) and slc-30A9 (yq158);slc-25A25 (yq373) were obtained by ethyl methanesulfonate (EMS) mutagenesis. slc-25A25 (yq406) mutants were generated by the CRISPR/Cas9 system. zcIs13 (P_hsp-6GFP) was provided by the Caenorhabditis Genetics Center (University of Minnesota, Minneapolis, MN). The drp-1 (tm1108) deletion mutant was provided by S. Mitani (Tokyo Women’s Medical University, Tokyo, Japan). The mcu-1 (ju1154) deletion mutant was obtained from S. H. Xu (Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, China). The integrated array hqIs181 (P_sdhl-1mtLs::GFP) was provided by M. Dong (National Institute of Biological Sciences, Beijing, China). The integrated arrays yqIs157 (P_Y37A1B.5mito-GFP) and yqIs179 (P_Y37A1B.5tomm-20::mCherry) were generated in this laboratory. Integrated arrays, deletion strains, and mutants generated by EMS mutagenesis or CRISPR/Cas9 were outcrossed with the N2 strain at least four times.

Extrachromosomal transgenes were generated by using standard microinjection methods. ≥3 independent transgenic lines were analyzed per construct. Transgenes used in this study are: yqEx1196 (P_F54A3.5F54A3.5::GFP), yqEx1193 (P_col-19slc-30A9::GFP), yqEx1195 (P_slc-30A9slc-30A9), yqEx1296 (P_slc-30A9slc-30A9(W305stop)), yqEx1271 (P_slc-30A9GFP), yqEx1030 (P_col-19slc-30A9(D198A;D202A)::GFP), yqEx1342 (P_col-19slc-30A9(D221A)::GFP), yqEx1345 (P_col-19slc-30A9(H402A)::GFP), yqEx1349 (P_Y37A1B.5slc-25A25::mCherry), yqEx1352 (P_Y37A1B.5slc-25A25::GFP), yqEx1515 (P_slc-25A25slc-25A25::GFP), yqEx1516 (P_slc-25A25hSLC25A25::GFP), yqEx1517 (P_tram-1tram-1::BFP), yqEx1518 (P_Y37A1B.5Mito-eCALWY-4), yqEx1519 (P_Y37A1B.5ER-eCALWY-4).

Expression constructs

Bacterial, C. elegans, and mammalian expression constructs are listed in Table S1.

RNA interference (RNAi)

RNAi experiments were performed by using the standard feeding method. For most experiments, 3–5 L4 larvae (P0) were placed on plates seeded with bacteria expressing gene-specific double-strand RNA and cultured at 20 °C. The F1 progeny at the L4 stage were transferred to fresh RNAi plates or control RNAi plates. The adults after L4 (F1) 24–48 h were used for further analysis. For slc-30A5 RNAi, 20 L4 larvae were placed on control (ctrl) RNAi and slc-30A5 RNAi plates and cultured at 20 °C. 24 h later, the adults were removed. Embryos were then analyzed for viability or developmental time.

EMS screening and gene cloning

Synchronized L4-stage animals were treated with 50 mmol/L EMS for 4 h. The F2 progeny at 24–48 h after the L4 molt were examined for phenotypes of interest. yq158, 166, 172, 189 and 212 were isolated from a screen of about 10,000 haploid genomes. yq158 was mapped to the genetic position from −12 to −10.69 on linkage group III (LGIII) using SNP mapping. The underlying mutations of all the mutants were identified with genome sequencing.

For the slc-30A9 (yq158) suppressor screen, each of about 3,000 haploid genomes were screened for suppressors of mitochondrial enlargement and for suppressors of UPR^mt (P_hsp-6GFP) signals. Chromosome mapping and gene identification were performed as above.

CRISPR/Cas9-mediated gene editing

To generate slc-25A25 (yq406) mutants, a single-guide RNA (sgRNA) targeting sequence (5′-CTGAATCTCCGTCGCTACG-3′) in the first exon of the slc-25A25 gene was cloned into the pDD162 vector, which expresses the Cas9 enzyme. A repair template containing the mutation of interest was designed to remove the cleavage site. A restriction site (Nhe I) was introduced into the repair template. dpy-10 was used as a positive control marker as described previously (Paix et al., 2014). pDD162 containing the sgRNA sequence (20 ng/μL) and repair templates (2 μmol/L) for the target gene were coinjected with the dyp-10 sgRNA construct (20 ng/µL) into gonads of young adult animals. Roller F1 worms were singled into new NGM plates, and the F2 progeny were examined by PCR amplification and restriction digestion. All mutations were confirmed by sequencing.

Predication of protein structure

A structural model of the C. elegans SLC-30A9 protein (residues 152–495) was predicted using the Phyre2 protein fold recognition server (Kelley et al., 2015) (template PDB accession No. 3J1Z). Extremely conserved residues that could be confidently predicted to coordinate Zn²⁺ were labeled and shown as sticks. Zinc ions were shown as magenta spheres. The picture was prepared using PyMOL (The PyMOL Molecular Graphics System, version 1.8.0; Schrödinger).

Cell culture, transfection, and reagents

HEK293 and HeLa cells were cultured at 37 °C with 5% CO₂ in DMEM (Gibco) with 10% heat-inactivated FBS (BioInd), 100 U/mL penicillin, and 100 µg/mL streptomycin. Transient transfections were performed with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. MitoTracker Deep Red (Thermo) staining was used to indicate the location of mitochondria in live cells.

Small RNA interference (siRNA)

Human SLC30A5/ZnT5 siRNA oligos used in the study were siRNA1: 5′-GCUGGAGUAAACAAUUUAATT-3′, siRNA2: 5′-GGCUGAUAGUAAACCUUAUTT-3′, siRNA3: 5′-GUGGUAGUGAGUGCUAUAUTT-3′; Control siRNA: 5′-UUCUCCGAACGUGUCACGUTT-3′.

Mixed oligos of siRNA 1, 2 and 3 (100 pmol for each) or control siRNA were transfected into HeLa cells using Lipofectamine 2000 (Invitrogen). 48 h after transfection, cells were observed under fluorescence microscopy or harvested for qPCR to evaluate the knockdown of SLC30A5/ZnT5. qPCR oligos for examining SLC30A5/ZnT5 expression were Forward: 5′-TTCTCCTATGGGTACGGCCGAAT-3′, Reverse: 5′-AGCCCTCCAACTGAGACTGGTGTT-3′.

Microscopy and imaging analysis

Differential interference contrast (DIC) and fluorescence images were taken by an inverted confocal microscope (LSM880; Carl Zeiss) using a 100× 1.46-NA oil objective lens. Imaging was performed on animals of the same developmental stage and on a similar body region of each animal, unless otherwise indicated. Images were processed and analyzed with ZEN 2 blue software (Carl Zeiss) or Image J (National Institutes of Health). All images were taken at 20 °C.

TPEN treatment of worms

Synchronized L4-stage worms were grown on OP50-seeded NGM plate containing 50 µmol/L or 100 µmol/L TPEN (N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine, Sigma) in at 20 °C. 24 h later, the mitochondrial morphology of animals were examined with confocal microscopy.

MitoTracker Red CMXRos and Zinpyr-1 treatment

For MitoTracker Red CMXRos staining in C. elegans, the synchronized animals (24 h after L4 molt) were soaked in 50 µL MitoTracker Red CMXRos (Invitrogen, 10 µmol/L in M9 buffer) for 10 min at 20 °C in the dark. The worms were then transferred to a new OP50-seeded NGM plate for 2 h at 20 °C in the dark and examined by fluorescence microscopy.

To visualize mitochondrial Zn²⁺ in HeLa cells, the cell-permeable fluorogenic Zn²⁺ reporter Zinpyr-1 (ChemCruz Biochemicals) was used by following the manufacturer's instructions. Briefly, a 1 mmol/L stock solution of Zinpyr-1 was diluted to a final concentration of 10 μmol/L in calcium- and magnesium-free PBS buffer. Before treatment, cells were washed by calcium- and magnesium-free PBS buffer 3 times, and then incubated with the diluted Zinpyr-1 solution for 20 min at 37 °C with 5% CO₂. After extensive washing, imaging was performed with fluorescence microscopy.

ATP measurements

500 synchronized animals (24 h after L4 molt) were picked to a tube contain 100 µL lysis buffer (ATP-Lite Assay kit; Vigorous). The mixture was frozen in liquid nitrogen for 5 min and then heated at 100 °C for 15 min. The mixture was cleared at 5,000 rpm for 5 min at 4 °C and the supernatant was applied to the luciferin-luciferase assays in the ATP-Lite Assay kit.

Life span assays

L4-stage worms were picked to fresh NGM plates and transferred to new plates every day until reproduction ceased. Worms were scored as dead if they had no response to touches on the head or tail. The surviving animals were recorded every day. Three repeats were performed for every strain.

Bending frequency analysis

Worms at the same developmental stage were picked onto an uncoated NGM plate and allowed to crawl free from any adherent food. Worms were then transferred to another uncoated NGM plate and soaked in M9 buffer. The number of body bends generated in a 1 min time interval were scored by eye. A body bend was defined as a change in the direction along the longitudinal axis from the head to the tail. The animals were recorded individually with the investigator blind to the genetic status of the worms.

Transmission electron microscopy

Adult animals were fixed using a high-pressure freezer (EM ICE, Leica). Freeze-substitution was carried out in anhydrous acetone containing 1% osmium tetroxide and 0.1% uranyl acetate dihydrate. The samples were sequentially placed at −90 °C for 72 h, −60 °C for 10 h and −30 °C for 10 h, and 0 °C for 5 h in a freeze-substitution unit EM AFS2 (Leica, Germany). After 3 washes (20 min each) with fresh cold anhydrous acetone, the samples were infiltrated with Embed-812 resin. Samples were embedded at 60 °C for 48 h and cut into 70 nm sections with a microtome (EM UC7, Leica, Germany). After electron staining with uranyl acetate and lead citrate, sections were observed with a HT7800 TEM (HITACHI, Japan) operating at 80 kV.

Recombinant proteins

cDNAs of interest were fused with the open reading frame of EGFP and cloned into the pGEX-4T1 vector and transformed into the E. coli Rosetta (DE3) strain. The bacteria were first grown at 37 °C to an OD₆₀₀ of 0.6 and then added with 0.8 mmol/L IPTG (isopropyl b-D-1-thiogalactopyranoside). The bacteria were then grown at 18 °C for 24 h to allow for expression of soluble proteins. Recombinant proteins listed below were extracted in the extraction buffer containing 20 mmol/L Tris-HCl, pH 8.0, 150 mmol/L NaCl, 0.1% NP-40, 10% glycerol, 1 mmol/L PMSF (phenylmethylsulfonyl fluoride) (Roche) and purified with Glutathione Sepharose 4B beads (GE Healthcare) according to the manufacturer’s instructions. GST-fusion proteins immobilized on the beads were eluted with Thrombin (Solarbio) in the protein buffer (25 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl) for 6 h at 37 °C to obtain recombinant proteins without the GST tag. Recombinant proteins tagged with EGFP were used for the MST assays, including: the Control protein, EGFP. C. elegans protein, SLC-25A25-EGFP. Human protein, SLC25A25-EGFP.

Isolation and purification of C. elegans mitochondria and Zn²⁺ measurement

Isolation and purification of C. elegans mitochondria were essentially performed as described previously (Li et al., 2009). Briefly, young adult worms obtained by liquid culture were collected and cleaned with 0.1 mol/L NaCl, followed by centrifugation in 30% sucrose solution at 3,500 rpm for 5 min at 4 °C The upper layer containing the adult worms was transferred to fresh solution containing 0.1 mol/L NaCl for further cleaning. Worms were then disrupted with ultra-sonication and subjected to differential centrifugation to obtain the crude mitochondria fraction. Mitochondria were purified with sucrose density gradient centrifugation using 55%, 40% and 30% sucrose gradients and 135,000 ×g at 4 °C for 2 h. The mitochondrial layer was collected and further washed, and examined by western blotting using antibodies against mitochondrial, lysosomal, and ribosomal proteins (provided by Dr. Xiaochen Wang, Institute of Biophysics, Chinese Academy of Sciences).

Mitochondrial contents were released in RIPA buffer and analyzed with ICP-MS (Inductively Coupled Plasma Mass Spectrometry) in the Toxicology Department of Peking University School of Medicine and Tsinghua University.

Microscale thermophoresis (MST) assays

To examine the binding of divalent cations with purified recombinant proteins, proteins at 200 nmol/L were incubated with varying concentrations of CaCl₂ and ZnSO₄ in a 10 µL mixture and loaded into NT.115 standard coated capillaries (NanoTemper Technologies). The buffers (25 mmol/L HEPES, pH 7.4, 150 mmol/L NaCl) used in the MST assays were free from EDTA/EGTA. MST measurements were performed at 25 °C, 60% excitation power, and medium MST power. All experiments were repeated three times for each measurement. Data analyses were performed using NanoTemper software.

To examine cation binding with EGFP-fused proteins expressed in HEK293 cells, 10 µL of cell lysates containing the protein of interest in lysis buffer (25 mmol/L Tris-HCl, pH 7.5, 100 mmol/L NaCl, 1% NP-40, 1% glycerol, 1 mmol/L protease inhibitor cocktail-EGTA free, and 1 mmol/L PMSF) were incubated with varying concentrations of divalent cations (ZnSO₄, CaCl₂, MgSO₄, CuSO₄, MnCl₂) and loaded into NT.115 standard coated capillaries. MST measurements and analysis were performed as above.

Fluorescence resonance energy transfer (FRET) imaging

The C. elegans Mito-eCALWY-4 construct was made by fusing 4 repeats of the mitochondrial targeting sequence of human cytochrome C oxidase subunit VIII (Cox VIII) to the N-terminus of eCALWY-4 and cloning into the pPD49.26 vector (Fig. 2G). The C. elegans ER-eCALWY-4 construct was generated by fusing the preproinsulin (PPI) sequence and the Lys-Asp-Glu-Leu (KDEL) sequence at the N- and C-terminus of eCALWY-4, respectively (Fig. 2G). Expression of both probes was controlled by the C. elegans hypodermal Y37A1B.5 promoter. Mito- and ER-eCALWY-4-expressing constructs were microinjected into N2 animals to obtain the extrachromosomal transgenic lines yqEx1518 (P_Y37A1B.5Mito-eCALWY-4) and yqEx1519 (P_Y37A1B.5ER-eCALWY-4). All strains expressing Mito-eCALWY-4 or ER-eCALWY-4 probes were obtained by introducing these extrachromosomal arrays.

Synchronized animals (24 h after L4 molt) were imaged using a confocal laser scanning microscope (LSM880; Carl Zeiss) with a 100× 1.46-NA oil objective lens. Cerulean (CFP) and Citrine (YFP) were excited at 458 and 514 nm, respectively, and the fluorescence emission was detected at 450–490 and 520–560 nm, respectively. The FRET channel was excited at 458 nm and fluorescence emission was detected at 520–560 nm. Normalized measure of FRET (FRETN) was performed as previously described (Gordon et al., 1998).

Fluorescence lifetime imaging microscopy (FLIM)-FRET

FLIM-FRET imaging for eCALWY-4 probes was performed on a Leica TCS SP8X scanning confocal microscope equipped with a 63×/1.3-NA water immersion objective, a white light laser (WLL), a HyD SMD single molecule detector and a 405-nm pulsed-laser excitation source. The cerulean donor (CFP) was excited by a 405-nm picosecond pulsed diode laser tuned at 40 MHz. Emitted photons passing through the 460–500-nm emission filter were detected using the HyD detector. The pixel frame size was set to 512 × 512, speed for 200, fluorescence lifetime frame repetition was acquired over 20, and the maximal photon counting rate was around 1 × 10⁶ counts. The fluorescence lifetime was analyzed using the Leica FALCON FLIM integrated software. “τ_c” indicates the fluorescence lifetime of the CFP donor.

Statistical analysis

The two-tailed unpaired Student's t test was used to ascertain statistically significant differences between two groups. One-way ANOVA with a Newman-Keuls post-test was used to ascertain statistically significant differences between multiple groups. For all quantifications, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant. Data were analyzed with GraphPad Prism Software (8.0.1) to generate curves or bar graphics with SEM (standard error of the mean) as y-axis error bars. Data distribution was assumed to be normal but this was not formally tested.

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Acknowledgements

This work was supported by grants from the National Science Foundation of China (91954204 and 31730053), the National Basic Research Program of China (2017YFA0503403), and Yunnan Province Science and Technology Department (#202001BB050077 and #202105AB160003). C. Yang is supported by Program of Yunnan Province Leading Talents in Science and Technology. We thank Dr. I. Hanson for proofreading the manuscript; Dr. G. Rutter (Imperial College of London) for providing the eCALWY-4 constructs; D. X. Wang (IBP, CAS) for providing C. elegans protein antibodies; Dr. L. Chen (Tsinghua Univ.) for ICP-MS analysis of mitochondrial divalent ions in HeLa cells; Dr. X. Ma (Zhengzhou Univ.) for help with SLC-30A9 structure prediction; and Dr. S. Xu (Zhejiang Univ.) as well as the Caenorhabditis Genetic Center (CGC) for C. elegans strains.

ABBREVIATIONS

AD, Alzheimer’s disease; APCs, ATP-Mg/phosphate carriers; CGC, Caenorhabditis Genetic Center; DIC, differential interference contrast; EGR1, early growth response factor 1; ERK1/2, extracellular signal-regulated kinase 1/2; ER, endoplasmic reticulum; EFhs, EF-hands; EMS, ethyl methanesulfonate; FRET, fluorescence resonance energy transfer; FLIM, Fluorescence Life-time Imaging Microscopy; HCC, hepatocellular carcinoma; ICP-MS, inductively coupled plasma mass spectrometry; MTs, metallothioneins; MMP, mitochondrial membrane potential; MPTP, mitochondrial permeability transition pore; MST, microscale thermophoresis; MCU, mitochondrial calcium uniporter; MST, Microscale thermophoresis; NGF, nerve growth factor; OXPHOS, oxidative phosphorylation; PCa, prostate cancer; RNAi, RNA interference; SNP, single nucleotide polymorphism; siRNA, small RNA interference; TRPP2, transient receptor potential channel polycistin-2; TEM, transmission electron microscopy; TPEN, N,N,N',N'-Tetrakis (2-pyridylmethyl) ethylenediamine; TCA, tricarboxylic acid; UPR^mt, mitochondrial unfolded protein responses; ZIPs, Zrt, irt-related proteins; Znt, Zn²⁺ transporters

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Tengfei Ma, Liyuan Zhao, Jie Zhang, and Ruofeng Tang have contributed equally to this work.

Authors and Affiliations

State Key Laboratory of Conservation and Utilization of Bio-resources in Yunnan and Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, 650091, China
Tengfei Ma, Liyuan Zhao, Jie Zhang, Ruofeng Tang, Xin Wang, Nan Liu, Qian Zhang, Fengyang Wang, Meijiao Li, Qian Shan, Yang Yang, Qiuyuan Yin, Limei Yang, Qiwen Gan & Chonglin Yang
State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China
Liyuan Zhao, Ruofeng Tang, Qian Zhang, Fengyang Wang & Qiwen Gan

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Tengfei Ma
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Liyuan Zhao
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Jie Zhang
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Ruofeng Tang
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Xin Wang
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Nan Liu
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Qian Zhang
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Fengyang Wang
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Meijiao Li
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Qian Shan
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Yang Yang
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Qiuyuan Yin
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Limei Yang
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Qiwen Gan
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Chonglin Yang
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Contributions

T. Ma, L. Zhao, J. Zhang, and R. Tang performed most of the experiments and interpreted the data. X. Wang and M. Li performed TEM analysis. N. Liu, F. Wang, Q. Zhang, Q. Shan, Q. Yin, Y. Yang and Q. Gan contributed to the experiments and materials. T. Ma, J. Zhang and C. Yang prepared the manuscript with discussion between all authors. C. Yang designed and supervised the study.

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Correspondence to Jie Zhang or Chonglin Yang.

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Ma, T., Zhao, L., Zhang, J. et al. A pair of transporters controls mitochondrial Zn²⁺ levels to maintain mitochondrial homeostasis. Protein Cell 13, 180–202 (2022). https://doi.org/10.1007/s13238-021-00881-4

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Received: 26 July 2021
Accepted: 02 September 2021
Published: 23 October 2021
Issue Date: March 2022
DOI: https://doi.org/10.1007/s13238-021-00881-4

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A pair of transporters controls mitochondrial Zn2+ levels to maintain mitochondrial homeostasis

Abstract

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INTRODUCTION

RESULTS

Loss of slc-30A9 leads to mitochondrial abnormality

SLC-30A9 is a mitochondrial Zn2+ exporter

Loss of slc-30A9 impairs mitochondrial function, compromises animal development and shortens the life span

Mutations in slc-25A25 suppress the abnormal mitochondrial enlargement in slc-30A9 mutants

SLC-25A25 is important for normal mitochondrial structure and function

SLC-25A25 binds with Zn2+ and is important for mitochondrial Zn2+ accumulation in slc-30A9 mutants

SLC-25A25/SLC25A25 overexpression leads to mitochondrial enlargement and Zn2+ elevation

Inhibiting the ER-localized Zn2+ transporter SLC-30A5 suppresses the mitochondrial Zn2+ accumulation in slc-30A9 mutants

ZnT5 inactivation ameliorates SLC-25A25-related mitochondrial defects

DISCUSSION

MATERIALS AND METHODS

C. elegans strains and genetics

Expression constructs

RNA interference (RNAi)

EMS screening and gene cloning

CRISPR/Cas9-mediated gene editing

Predication of protein structure

Cell culture, transfection, and reagents

Small RNA interference (siRNA)

Microscopy and imaging analysis

TPEN treatment of worms

MitoTracker Red CMXRos and Zinpyr-1 treatment

ATP measurements

Life span assays

Bending frequency analysis

Transmission electron microscopy

Recombinant proteins

Isolation and purification of C. elegans mitochondria and Zn2+ measurement

Microscale thermophoresis (MST) assays

Fluorescence resonance energy transfer (FRET) imaging

Fluorescence lifetime imaging microscopy (FLIM)-FRET

Statistical analysis

References

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Supplementary Information

Supplementary file1 (PDF 154177 kb)

Supplementary file2 (XLSX 12 kb)

Supplementary file3 (XLSX 11 kb)

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A pair of transporters controls mitochondrial Zn²⁺ levels to maintain mitochondrial homeostasis

SLC-30A9 is a mitochondrial Zn²⁺ exporter

SLC-25A25 binds with Zn²⁺ and is important for mitochondrial Zn²⁺ accumulation in slc-30A9 mutants

SLC-25A25/SLC25A25 overexpression leads to mitochondrial enlargement and Zn²⁺ elevation

Inhibiting the ER-localized Zn²⁺ transporter SLC-30A5 suppresses the mitochondrial Zn²⁺ accumulation in slc-30A9 mutants

Isolation and purification of C. elegans mitochondria and Zn²⁺ measurement