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Human Molecular Genetics Advance Access originally published online on July 2, 2007
Human Molecular Genetics 2007 16(17):2061-2071; doi:10.1093/hmg/ddm154
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Inverse correlation between expression of the Wolfs Hirschhorn candidate gene Letm1 and mitochondrial volume in C. elegans and in mammalian cells

Ayako Hasegawa and Alexander M. van der Bliek*

1 Department of Biological Chemistry, David Geffen School of Medicine, UCLA, Box 951737, Los Angeles, CA 90095 USA

* To whom correspondence should be addressed. Tel: +1 3108259779; Fax: +1 3102065272; Email: avan{at}mednet.ucla.edu

Received May 9, 2007; Revised June 12, 2007; Accepted June 15, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Deletion of the Letm1 gene correlates with the occurrence of epilepsy in patients with Wolf–Hirschhorn syndrome. The Letm1 gene encodes a mitochondrial protein that is homologous to yeast Mdm38. Yeast Mdm38 is localized to the mitochondrial inner membrane where it was proposed to act as a K+/H+ antiporter or alternatively as a chaperone for selected mitochondrial inner membrane proteins. Here, we present cellular and biochemical analysis of Letm1 in mammalian cells and an analysis of a C. elegans mutant that could serve as a model for Wolf–Hirschhorn syndrome. We localized the Letm1 protein to the mitochondrial inner membrane of mammalian cells, where it exists in a 550-kDa complex. We show that Letm1 can bind to itself in vitro, raising the possibility that it can form higher order multimers in vivo. Reduced levels of Letm1 in human cells and in C. elegans lead to swellings along the lengths of mitochondria, consistent with the phenotype observed in yeast. Electron micrographs show mitochondria with swollen matrices that are less electron-dense than matrices in normal mitochondria. The opposite effect is achieved by overexpression of Letm1. Overexpression increases the electron density of the mitochondrial matrix and swelling of cristae. Our results are therefore consistent with a protein that regulates the volume of the mitochondrial matrix.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Letm1 gene was first identified as one of several genes within a chromosomal interval that is deleted in patients with Wolf–Hirschhorn syndrome (1). Patients with this syndrome can have growth delays, mental retardation, epileptic seizures and they can have facial abnormalities, including facial closure defects (2). Differences in the combinations of symptoms and their severity are thought to result from the extent of the chromosomal deletion in individual patients (3). Although the gene or genes that are responsible for the observed pathologies have not yet been positively identified, Letm1 is considered a strong candidate for the epileptic component, because it is a mitochondrial protein and mitochondrial defects often have wide-ranging and varied consequences including epilepsy (4). Moreover, deletion of the Letm1 gene correlates well with the occurrence of epilepsy in Wolf–Hirschhorn syndrome patients (5).

The yeast homologue of Letm1 was identified in a visual screen for mutants with mitochondrial morphology defects (6). Mutations in this protein, which is called Mdm38 in yeast, cause swelling of the mitochondria and disruption of their cristae. Mutations in Mdm38 also suppress certain mitochondrial splicing defects that were suggestive of an ion imbalance (7). Further experiments suggest that loss of Mdm38 specifically affects mitochondrial potassium homeostasis by acting as part of a K+/H+ antiporter or by regulating its activity (8,9). Mitochondrial K+/H+ antiporters utilize the proton gradient generated by oxidative phosphorylation to remove potassium from the mitochondrial matrix. Removal of potassium causes efflux of water, thereby increasing the protein concentration within the mitochondrial matrix. More direct evidence favoring a role of Letm1 in potassium homeostasis comes from measurements of ion concentrations and ion flux in mitochondria isolated form wild-type and from Mdm38 mutant yeast (8). These experiments show a direct correlation between the presence of Mdm38 and electro-physiological measurements of K+/H+ antiporter activity.

Mdm38 was also shown to affect mitochondrial protein export (10). Mitochondrial protein export encompasses targeting and maturation of mitochondrial proteins that are encoded by mitochondrial DNA, synthesized within the mitochondrial matrix and cotranslationally inserted into the mitochondrial inner membrane (11). Similar to the previously described chaperone Oxa1 (11), Mdm38 was shown to bind mitochondrial ribosomes, while mutations in Mdm38 markedly affected the transport of two proteins encoded by mitochondrial DNA (ATP6 and cytochrome b) (10). Their disruption causes secondary defects in the assembly of respiratory complexes. It is possible that Mdm38 is dual functional, mediating both potassium efflux and protein export, but it seems more likely that Mdm38 is directly linked to only one of these two processes, while other effects of mutations in Mdm38 might be indirect. Knowing which of these effects are direct and which are indirect may help reveal the function of Mdm38.

The sequence of Letm1 does not give clues to the function of the protein. It has an amino-terminal mitochondrial targeting sequence that is cleaved upon import into mitochondria. The next 30 amino acids are poorly conserved, but do contain a predicted coiled coil. This segment is followed by a domain of about 270 amino acids, which is highly conserved in eukaryotes with mitochondria, but not in amitochondriate protists (Trichmonas, Entamoeba and Giardia) or in bacteria. This conserved domain includes a hydrophobic segment that is long enough to span the membrane once, but not multiple times as expected of an ion transporter. The remaining C-terminal sequences are poorly conserved, but do contain putative coiled coils. The EF hand motif that was originally noted in some Letm1 homologues is poorly conserved among species and when compared with other EF hand motifs, suggesting that Letm1 is not regulated by calcium (1,12).

Here, we present our analysis of Letm1 in mammalian cells and in C. elegans. With preparations made from mammalian cells we show that Letm1 exists in a 550-kDa complex embedded in the mitochondrial inner membrane. We analyzed gain and loss of function of Letm1 in mammalian cells and in C. elegans and we characterize a C. elegans Letm1 mutant that may serve as model for Wolf–Hirschhorn syndrome. In both organisms, decreased expression of Letm1 causes mitochondria to swell, whereas increased expression of Letm1 reduces mitochondrial membrane potential and causes the inner and outer membranes of mitochondria to separate. Electron microscopy shows that under and overexpression correspond with decreases and increases in electron density, suggesting that mitochondrial volume and protein concentrations are controlled by the levels of Letm1 expression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Localization of Letm1 in mammalian cells
The mitochondrial localization of endogenous Letm1 was previously shown with immunofluorescence (12). To confirm this result and to provide material for further biochemical analyses, we generated a polyclonal antibody against bacterially expressed Letm1 protein. Immunofluorescence of endogenous Letm1 protein (Fig. 1) and Letm1 with a C-terminal myc-tag (data not shown) both show colocalization with endogenous AIF and with Mitotracker. Our antibody recognizes a band of ~80 kDa when analyzed with 8% SDS–PAGE (data not shown). We expect the mature protein to be ~80 kDa, taking into account cleavage of the mitochondrial leader sequence as predicted by PSORT. The intensity of this band is greatly reduced in cells transfected with Letm1 siRNA oligonucleotides, confirming that it is indeed Letm1 (Fig. 5D). Together these results show that our antibody recognizes Letm1 and that this protein is indeed localized to mitochondria.


Figure 1
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  		Figure 1. Localization of Letm1 in mammalian cells. (A) Staining of a HEK 293T cell with one of our Letm1 antibodies. (B) The same cell stained with Mitotracker. (C) The merged image of Letm1 immunofluorescence (green) and Mitotracker staining (red) shows colocalization. Scale bar is 10 µm.

 
Submitochondrial localization of Letm1
Yeast Mdm38 was localized to the mitochondrial inner membrane with differential centrifugation, protease protection and carbonate extraction (9,10). We used similar approaches to test whether mammalian Letm1 is localized to the mitochondrial inner membrane. Mitochondria were isolated from HEK 293T cells and subjected to differential centrifugation. Western blotting shows that the Letm1 protein cofractionates with mitochondria (Fig. 2A), as expected from the immunofluorescence results. We then conducted protease protection experiments with mitochondria isolated from bovine brain as previously described (13). Isolated mitochondria were incubated with increasing concentrations of digitonin and trypsin. When no digitonin is added, Letm1 remains fully protected against digestion by trypsin, but when increasing amounts of digitonin are added, Letm1 becomes sensitive to trypsin digestion (Fig. 2B). This effect is most noticeable when comparing lanes with 0 and 25 µg/ml Trypsin. The amount of digitonin needed to achieve proteolysis is similar to the amount needed for digestion of a protein exposed to the mitochondrial inter membrane space (Tim23), while proteins in the mitochondrial matrix (GDH and Hsp60) remain protected from digestion, even at higher digitonin concentrations. These results indicate that Letm1 is at least partially exposed to the mitochondrial intermembrane space. We also conducted carbonate washes to determine whether Letm1 is an integral membrane protein. The results show that Letm1 remains in the pelleted fraction when incubated with carbonate at pH 13, while a peripheral membrane protein (ATP synthase {alpha} subunit) is released into the supernatant (Fig. 2C). We conclude that Letm1, like its yeast counterpart Mdm38 (9,10), is an integral membrane protein localized to the mitochondrial inner membrane.


Figure 2
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  		Figure 2. Biochemical characterization of Letm1 in mammalian cells. (A) Letm1 is largely in the medium speed pellet (P2), along with the marker for mitochondria (porin), while the marker for cytoplasmic proteins (tubulin) is in the supernatant (S2). Letm1 also cofractionates with porin in the Percoll gradient (only the relevant fraction containing mitochondria is shown). The homogenate (H), S1 and S2 lanes contain volume equivalents, while the P2 lane was 10 x concentrated and the Percoll fraction was 60 x concentrated. (B) Submitochondrial localization was determined with a protease protection experiment. Isolated mitochondria were subjected to increasing amounts of digitonin and increasing concentrations of Trypsin as indicated. These samples were analyzed by probing western blots with Letm1 antibody and antibodies for a protein exposed to the mitochondrial intermembrane space (Tim23) as well as antibodies for mitochondrial matrix proteins (GDH and Hsp60). (C) Extraction with carbonate pH13 shows that Letm1 is an integral membrane protein. Most if not all Letm1 is in the membrane pellet (P), together with a marker for membrane proteins (Tim23), while a marker for soluble proteins (ATP synthase {alpha} subunit) is in the supernatant (S).

 
Size of the Letm1 protein complex
Letm1 has a number of coiled-coil forming segments, suggesting that it may form homo-multimers or associates with other proteins. We tested whether Letm1 can form homo-multimers using coIPs of GFP and HA tagged proteins. Our results show that Letm1 can indeed bind to itself in vivo (Fig. 3A). To investigate whether Letm1 is in a complex that might form a channel, we determined the size of a mammalian Letm1-containing complex using size exclusion chromatography. Mitochondria were isolated from bovine liver and solubilized with 0.4% Triton X-100. We find that Letm1 elutes in a complex with an apparent molecular weight of ~550 kDa (Fig. 3B). We were unable to coIP other proteins along with Letm1, despite testing a range of lysis and labeling conditions (data not shown). Although it remains possible that Letm1 associates with other proteins, our data shows that it can form homomultimers and exists in a complex large enough to contain six or seven Letm1 subunits.


Figure 3
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  		Figure 3. Complex formation. (A) Formation of Letm1 dimers in vivo. HEK 293T cells were transfected with Letm1 fused to GFP, with Letm1 fused to 3 x HA tags or transfected with both constructs. Detergent lysates from these cells were immunoprecipitated with HA antibodies and eluted by boiling in 1 x Laemmli sample buffer. (B) Size of the Letm1 protein complex determined with size exclusion chromatography. Isolated mitochondria were lysed with 0.4% Triton X-100 and size fractionated with a Superose-12 gel filtration column. Samples were size-fractionated with SDS–PAGE and western blots were probed with Letm1 antibodies, as shown in the lower panel. The relative amount of Letm1 protein in each sample was determined by densitometry. The relative amounts are shown with open circles (%). The size of the Letm1 complex was determined by plotting elution profiles of the size markers (X) against that of Letm1.

 
Effects of reduced Letm1 expression in mammalian cells
To determine the effect of reduced Letm1 expression in mammalian cells, we transfected HeLa cells with Letm1 siRNAs (double strand RNA oligonucleotides matching positions 835 to 857 of the coding sequence). The Letm1 siRNA transfected cells were stained with Mitotracker. A few swollen mitochondria were observed 72 h after transfection, but at 96 h up to 43% of cells had swollen mitochondria (n = 120 cells, Fig. 4B). In contrast, only 7% of cells transfected with scrambled oligonucleotides had swollen mitochondria, indicating that the Letm1 siRNA effects were specific (n = 124 cells, Fig. 4A). Eventually, the cells transfected with Letm1 siRNA round up and became detached, suggesting that they are dying. There was, however, no obvious difference in the amount of Mitotracker staining, suggesting that the mitochondrial membrane potential was not affected.


Figure 4
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  		Figure 4. Effects of Letm1 knock down and over expression in mammalian cell culture. (A) Mock-treated Hela cells stained with Mitotracker were observed by light microscopy. Tubular mitochondria are observed throughout the cell. (B) Hela cells 96 h after treated with Letm1 siRNA shows large mitochondrial blebs that stain with Mitotracker. (C) Mitotracker staining of COS7 cells transfected with a Letm1 overexpression construct. (D) Transfected cells were identified with Tom20::GFP as a cotransfected marker. (E) Merged image with Mitotracker in red and Tom20::GFP in green. (F) Mitotracker staining of a wild-type human fibroblast and (G) Mitotracker staining of a fibroblast obtained from a patient with Wolf–Hirschhorn syndrome. The one shown here is GM00343, but two other patient fibroblast cell lines obtained from Corriell Cell Repositories also have mitochondria that look wild-type. Scale bar = 5 µm.

 
Effects of increased Letm1 expression in mammalian cells
To help understand Letm1 function, we also examined the effects of high levels of Letm1 expression. COS7 cells were transfected with a construct containing Letm1 cDNA under control of the CMV promoter. These cells were cotransfected with a Tom20::GFP construct to detect the mitochondrial outer membrane and they were stained with Mitotracker to detect the inner membrane. In almost all of the transfected mammalian cells, recognizable by their abnormal mitochondrial morphologies and the cotransfected marker, we find that staining with Mitotracker is much reduced in comparison with the staining in adjacent untransfected cells (Fig. 4C–E). This difference is also observed when cytosolic GFP is used as cotransfected marker, but not when Tom20::GFP is transfected alone (data not shown), demonstrating that this effect is due to Letm1 overexpression. The pronounced difference in staining shows that overexpression of Letm1 causes loss of mitochondrial membrane potential.

To determine whether the mitochondria of Wolf–Hirschhorn syndrome patients also have altered mitochondrial morphologies, we obtained fibroblasts from affected patients through the Coriell Cell Repositories. These cells were stained with Mitotracker and the results were compared with our siRNA results. We find that mitochondria in the patient fibroblasts were morphologically indistinguishable from mitochondria of normal fibroblasts: their diameters and distributions appeared to be unaffected unlike the mitochondria of Letm1 siRNA transfected cells (Fig. 4F–G). Although there is apparently enough Letm1 protein in these fibroblasts to sustain normal mitochondrial morphology, other cell types, such as neurons, may place a higher demand on mitochondria and could therefore be affected by partial loss of Letm1 function.

Effects of altered expression on the ultrastructure of mitochondria
To further examine the effects of Letm1 under- and overexpression, we prepared samples of cells transfected with siRNA or with the CMV expression construct for electron microscopy. The mitochondria of cells harvested at 96 h after transfection with Letm1 siRNA were swollen (covering 1.8 times the surface area of mitochondria in untransfected cells, as determined with electron micrographs) and they had fewer cristae than cells transfected with scrambled oligonucleotides (Fig. 5A and B). The mitochondrial matrices were less electron-dense than the matrices of mitochondria in control samples, suggesting that the swelling of mitochondria caused their proteins to be more dilute. The opposite effects were observed in mitochondria of cells transfected with the Letm1 expression construct. These mitochondria were smaller than those in mock transfected cells, the matrices were much more electron dense and the cristae were swollen (Fig. 5C). These characteristics are similar to those of mitochondria in the orthodox conformation, a conformation that is observed when isolated mitochondria are incubated with ADP or other metabolism inducing conditions.


Figure 5
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  		Figure 5. Ultrastructural effects of Letm1 knock down and over expression in Hela cells. (A) Mitochondria of Hela cells mock-treated with scrambled oligos show regular mitochondria with transverse cristae. (B) Mitochondria of Hela cells transfected with Letm1 siRNA are swollen and have fewer cristae. (C) Mitochondria of Hela cells transfected with a Letm1 expression construct have condensed matrices and swollen cristae. (D) Western blot of extracts from mock-transfected (lane 1) and Letm1 siRNA-transfected (lane 2) Hela cells. Dramatic reduction in Letm1 expression level was observed 96 h after siRNA treatment, while tubulin, which serves as a loading control, shows no difference. Scale bar = 1 µm.

 
Expression and localization of Letm1 in C. elegans
The C. elegans homologue of Letm1 is encoded by the F58G11.1 gene (79% amino acid identity between C. elegans and human proteins). The F58G11.1 gene (henceforth designated as letm-1) produces two splice variants that encode proteins with or without a 14 amino acid insertion near their C-termini. To determine the expression pattern of letm-1, worms were injected with a construct containing a 3-kb fragment encompassing the letm-1 gene promoter fused to a nuclear targeting signal and GFP. The highest levels of expression were detected in intestine cells, but we also observed expression in a wide range of other cell types (neurons, muscles and hypodermal cells) suggesting that letm-1 is ubiquitously expressed (data not shown). Subcellular localization of the protein was determined by injecting worms with a fusion construct encoding LETM-1::YFP under control of the muscle specific myo-3 promoter along with mito::CFP as a mitochondrial marker. Figure 6 shows colocalization of mito::CFP and LETM-1::YFP signals. It is therefore likely that C. elegans LETM-1 is a mitochondrial protein, similar to the homologous proteins in other species.


Figure 6
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  		Figure 6. Localization of LETM-1 in C. elegans. The images show a C. elegans body wall muscle expressing LETM-1::YFP (A, and green in C) and CFP targeted to the mitochondrial matrix (B, and red in C). The two labels colocalize, showing that LETM-1::YFP is targeted to mitochondria. The nucleus is marked with N. Scale bar = 5 µm.

 
Gross anatomical defects caused by Letm-1 loss of function in C. elegans
The National Bioresource Project in Japan isolated a mutant allele of C. elegans letm-1 called tm1234. This allele is most likely a null, since it has a deletion that causes a frameshift and a premature stop codon, giving rise to a protein with little more than the mitochondrial leader sequence. Because this allele could not be maintained as a homozygote, it was balanced with the chromosomal translocation nT1. A small number of homozygous letm-1(tm1234) progeny hatch and survive, but those invariably arrest at the L3 larval stage. These arrested animals can live longer than wild-type animals (some survive up to 43 days, compared with 23 days for N2 animals, see Fig. 7A), but they remain small and are unable to reproduce. In contrast, letm-1(tm1234) heterozygous animals are phenotypically indistinguishable from wild-type animals. Heterozygous C. elegans letm-1 mutants also show no signs of epilepsy, even when challenged with threshold levels of drugs such as pentylenetetrazole, 4-aminopyridine and 3-nitropropionic acid (data not shown) that can induce seizures in other C. elegans mutants (14). We conclude that the tm1234 allele is fully recessive and that the letm-1 gene is not haplo-insufficient in worms.


Figure 7
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  		Figure 7. The effects of letm-1 loss of function on growth and survival of C. elegans. (A) Survival of letm1 homozygous mutants and wild-type animals. Homozygous mutants (open circles) were recognized by lack of GFP (the balancer nT1 has a GFP marker) and tracked for extended periods of time. Survival of wild-type N2 animals (closed circles) is shown for comparison. (B) Lengths of worms that were grown from hatching on letm-1 feeding RNAi bacteria (open circles) or on bacteria with vector alone (closed circles). The lengths were determined by periodically photographing the worms on the plates. (C) A typical worm grown for 4 days at 20°C on bacteria containing the feeding RNAi vector without insert. This worm has reached adulthood and contains many fertilized eggs. (D) A worm grown for 4 days on bacteria containing the letm-1 feeding RNAi construct. This worm has reached adulthood, but is much smaller and contains fewer eggs than the control animal. Scale bar = 50 µm.

 
To look at intermediate phenotypes, we utilized a variety of RNAi approaches with which we could manipulate the severity of LETM-1 loss of function. C. elegans N2 animals (the wild-type strain) were grown on bacteria expressing dsRNA or microinjected with dsRNA. Control animals, which were fed with bacteria transformed with empty vector, reached adulthood within 3 days after hatching. Animals grown from hatching on bacteria with letm-1 dsRNA took 4 days to reach adulthood, but they stayed much smaller than control animals (Fig. 7B–D). The effect of letm-1 RNAi on brood size was determined with microinjected animals. Worms that were injected with letm-1 dsRNA have on average 80% smaller brood sizes than worms that were injected with solvent alone (mean = 38 and 208, SD = 26 and 43, n = 32 and 22, respectively). We conclude that the RNAi phenotypes are similar to those of letm-1(tm1234), but the onset and severity of these phenotypes depend on the timing and effectiveness of the methods used for RNAi.

Effects of altered Letm-1 expression on C. elegans mitochondria
The mitochondrial matrix was labeled with CFP fused to a mitochondrial leader sequence. The mitochondrial outer membrane was labeled with YFP fused to a small part of C. elegans Tom70 as described previously (15). Both fusion proteins were expressed with the muscle specific myo-3 promoter. Control animals, which were mock-injected with water, have tubular mitochondria, which are typically uniform in diameter (0.5–1 µm) and often run parallel to the body axis (Fig. 8A). In contrast, animals injected with letm-1 dsRNA have mitochondria that are highly disorganized and have large variations in their diameter (Fig. 8C). Many mitochondria are greatly enlarged (up to 5 µm in diameter), sometimes making them even larger than nuclei.


Figure 8
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  		Figure 8. Effects of altered LETM-1 expression on C. elegans mitochondria. (A) Tubular mitochondria of a control worm that was mock injected runs parallel to each other and its body axis. Green shows mitochondrial outer membrane targeted YFP. Red shows mitochondrial matrix targeted CFP. The nucleus is marked with N. (B) Mildly affected mitochondria of worms that were grown on letm-1 feeding RNAi bacteria. Mitochondria are mildly swollen and irregularly shaped. (C) Severely affected mitochondria in a worm that was microinjected with letm-1 dsRNA. This treatment leads to more severe loss of expression than achievable with feeding RNAi. Some mitochondrial blebs are swollen to the size of nucleus, other smaller mitochondrial blebs also appear distended. (D) Mitochondria of a transgenic worm with high levels of LETM-1 expression obtained with the myo-3 promoter. The mitochondrial outer membrane (MOM) is sometimes distended and separated from the matrix compartment (see inset). (E) Rescue of the mitochondrial defects caused by letm-1 feeding RNA. Rescue was achieved by expression of a GFP tagged version of C. briggsae letm-1 (green), which is insensitive to C. elegans letm-1 RNAi because of the many silent mutations that have occurred during the course of evolution. Scale bar = 5 µm.

 
To verify that the effects on growth and mitochondrial morphology were not due to inadvertent silencing of other genes, we performed a rescue experiment of worms fed with the RNAi bacteria. In this rescue experiment, C. briggsae letm-1 genomic DNA fused to GFP was expressed under control of the letm-1 promoter. The nucleotide sequences of C. briggsae genes have enough differences with C. elegans genes to avoid silencing. By looking at brood size (data not shown) and mitochondrial morphology (Fig. 8E) in the second generation after transferring to feeding RNAi bacteria, we were able to determine that the RNAi effects are specifically attributable to knock down of the letm-1 gene.

Feeding RNAi was used to obtain a milder phenotype. Animals fed from hatching with letm-1 dsRNA bacteria had mitochondria with slightly enlarged and somewhat irregular diameters (Fig. 8B). The mitochondria appeared to have indentations, suggestive of localized constraints on mitochondrial swelling. It is unclear whether these constraints come from within mitochondria or act on the outside of mitochondria. These milder defects in mitochondrial morphology are similar to those observed with drugs that inhibit oxidative phosphorylation, such as thenoyltrifluoroacetone, rotenone and antimycin, and they are similar to the morphologies observed with RNAi of a wide range of OxPhos components (16). To study the effects of increased LETM-1 expression, we placed full length LETM-1 encoding DNA under control of the myo-3 promoter. Animals made transgenic with this construct showed marked crimping of the mitochondrial matrix, while the outer membrane was sometimes swollen. In a few instances, it was apparent that the mitochondrial outer membrane detaches from the matrix compartment (Fig. 8D). We conclude that the levels of C. elegans LETM-1 are inversely correlated with the volume of the mitochondrial matrix, similar to effects of under and overexpression of Letm1 in mammalian cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Both C. elegans and mammalian cells show swelling and crimping of the mitochondrial matrix as the most striking features of Letm1 under and overexpression. Swelling of mitochondria caused by loss of Letm1 function is readily apparent in C. elegans where it is far more extreme than the mitochondrial abnormalities that are observed with reduced expression of Ox-Phos proteins (16). Swelling of mitochondrial matrices is also observed in mammalian cells transfected with Letm1 siRNA. The opposite effect, crimping of the mitochondrial matrix in cells that overexpress Letm1, is suggested by separation of the mitochondrial inner and outer membranes in C. elegans and by the increased electron density that is observed with electron microscopy of mammalian cells. We conclude that Letm1 is important for controlling the volume of the mitochondrial matrix.

Letm1 is also essential for growth and survival as shown by the lethality of a homozygous deletion of C. elegans letm-1. In agreement with this finding, none of the patients with Wolf–Hirschhorn syndrome are homozygous (3), nor do homozygous mice with large deletions covering the Wolf–Hirschhorn region survive embryogenesis (17). Mice that are heterozygous for these deletions have midline, craniofacial and ocular abnormalities and show signs of seizures, similar to the pathology in humans (17). The pathologies that occur in hemizygous humans and mice indicate that one or more genes in this region are haploinsufficient. Although these deletions are too large to determine unequivocally whether deletion of Letm1 contributes to epileptic seizures, it does correlate well with the occurrence of epilepsy in Wolfs–Hirschhorn patients (5).

The mitochondrial localization of the Letm1 protein is also consistent with a role in epilepsy, since several other forms of epilepsy are caused by mitochondrial defects (4) and the epileptic seizures of Wolf–Hirschhorn patients can be induced by fever, which is also consistent with a mitochondrial origin (18). Fevers are thought to cause non-specific proton leakage across the mitochondrial inner membrane, which disrupts mitochondrial function. If heterozygous mutations in the Letm1 gene indeed cause epilepsy in humans, then the ensuing defect in mitochondrial function must be subtle and may only affect the most sensitive tissues. Brain tissue might be more readily affected since it relies heavily on ATP produced by mitochondria, as shown by the sensitivity of brain function to levels of oxygen (19).

How might mitochondrial defects cause epilepsy? Mitochondria are best known for their role in ATP production, but they are also needed to buffer calcium and they play a critical role in cell death. Although any of these factors could, in principle, contribute to epileptic seizures, defects in ATP production may be the most important, since other mitochondrial defects that cause epilepsy, such as MERRF, are first and foremost defects in oxidative phosphorylation (4). Further evidence that ATP production by oxidative phosphorylation is a factor in epilepsy comes from the observation that epilepsy can be reversed by diet-induced ketosis (20). Reduced levels of ATP might affect the sodium/potassium ATPase at the plasma membrane (mutations in the sodium/potassium ATPase also cause epilepsy) (21). Reduced electrical potential at the plasma membrane is thought to enhance the sensitivity of neurons to glutamate, which may lead to the hyperexcitability that occurs in epileptic seizures (22).

The two proposed functions of Letm1 (K+/H+ exchange or insertion of OX-Phos components into the inner membrane) are both likely to affect ATP production. The C. elegans letm-1 mutants also show strong growth and mitochondrial morphology defects that are consistent with reduced levels of ATP. The finding that C. elegans heterozygotes are normal could reflect physiological differences between C. elegans and humans. There is at least one other neurological defect caused by haploinsufficiency of a mitochondrial protein in humans that is fully recessive in C. elegans. Hemizygous mutations in the Opa1 locus cause dominant optic atrophy in humans (23), while hemizgous deletions in the C. elegans counterpart eat-3 are wild-type (unpublished data). More definitive evidence for Letm1 haplo-insufficiency in mammals will require targeted mutagenesis of the mouse Letm1 gene or identification of a spontaneous mutation in the human Letm1 gene without mutations in surrounding genes.

The idea that yeast Mdm38 is involved in mitochondrial protein export was based in part on coimmunoprecipitation of Mdm38 with ribosomal subunits and components of the Ox Phos machinery (10). We were unable to coprecipitate Ox-Phos components with Letm1 from mammalian cells (data not shown), but this difference may have resulted from different solubilization conditions or different pull down conditions. The weakest effects observed with loss of Letm1 function in C. elegans are similar to the effects observed with RNAi of OX-Phos components. However, the extreme swelling of mitochondria that resulted from strong loss of Letm1 function was never observed with RNAi of OX-Phos components, nor was it observed with chemical inhibitors of OX-Phos. Extreme swelling of mitochondria is more consistent with a K+/H+ imbalance. Valinomycin has a similar effect. Although overexpression sometimes causes side effects that are unrelated to the primary function of a protein, we find that overexpression of Letm1 has an effect that is opposite to the effects of loss of function. We conclude that Letm1 expression levels are inversely correlated with the volume of the mitochondrial matrix, a phenotype that is more consistent with control of K+/H+ exchange than with mistargeting of OX-Phos components.

The amino acid sequence of Letm1 has only one hydrophobic segment and this sequence is interrupted by one or two charged residues, raising the question how the protein is anchored in the inner membrane. Protease protection experiments done with yeast Mdm38 show protection under conditions that expose intermembrane space proteins to protease digestion, suggesting that both N-terminal and C-terminal face the mitochondrial matrix (9,10). In contrast, our results with Letm1 in mammalian mitochondria show at least some protease sensitivity under conditions that selectively expose intermembrane space proteins. This difference might have been caused by differences in intrinsic protease sensitivity or the use of different approaches (hypotonic swelling is used to expose the intermembrane space in yeast protease protection experiments, while permeabilization with digitonin is used for mammalian experiments) (24). These data could be more easily reconciled if Letm1 is anchored with a hairpin loop, similar to caveolin, DP1 and Rtn4c/NogoC (25), with the bulk of the protein facing the matrix but at least in mammals the loop would be partially exposed to the mitochondrial intermembrane space. Either way, a single Letm1 molecule will not have enough transmembrane segments to transport ions across the membrane. Our biochemical data does show that Letm1 can dimerize and that it is in a 550-kDa complex, which is consistent with six or seven Letm1 subunits or an association with other as yet unidentified proteins. Biochemical reconstitution will be needed to determine unequivocally whether the larger complex containing Letm1 mediates K+/H+ exchange directly or has an indirect effect on exchange.

The critical role of mitochondrial potassium flux for cell survival is underscored by cardiac diseases resulting from mitochondrial potassium imbalance (26). Some of these may be due to defects in K+/H+ exchange, but others result from defects in an ATP gated potassium channel in mitochondria. Although this channel has been characterized more extensively, its identity is not yet known (27). However, two other channels, which normally are localized to the plasma membrane are also localized to the mitochondrial inner membrane in some cell types. One of these is a large conductance calcium-activated potassium channel (28), while the other is the voltage gated potassium channel Kv1.3, which was localized to mitochondria in lymphocytes (29). These genes are not part of the Wolf–Hirschhorn syndrome locus, but the presence of the corresponding proteins in mitochondria shows that mitochondria go to great lengths to regulate potassium flux. Our work with Letm1 in worms and human cells lays the necessary foundation for future research in this field, which is all the more important since the role of Letm1 in potassium flux and its contribution to epilepsy are in question.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cloning and expression of C. elegans DNAs
C. elegans genomic DNA was prepared using standard procedures (15). A genomic fragment containing the C. elegans letm-1 gene promoter was obtained by PCR from genomic DNA from the Bristol N2 strain. The pPD expression vectors were kindly provided by A. Fire, J. Ahnn, G. Seydoux and S. Xu (Carnegie Institution of Washington, Baltimore, MD). The Pletm-1::NLS::GFP::ß-galactosidase construct was made with a letm-1 gene promoter fragment (positions 13659910–13662874 of chromosome V), fused to the reporter sequences of pPD96.04. The letm-1 coding region was expressed in muscle cells using the myo-3 promoter of pPD96.52. The antisense construct has DNA from position 1–1221 of F58G11.1a genomic DNA cloned in the antisense orientation in pPD96.52.

Common C. elegans and C. briggsae strains were obtained from the C. elegans stock-center (CGC, University of Minnesota). C. elegans letm-1(tm1234) animals were kindly provided by Dr K. Mitani (Tokyo, Japan). The mitochondrial markers, injection, RNAi and imaging procedures were described previously (15,30). The expression constructs were injected along with the transformation marker, rol-6(su1006), into wild-type C. elegans (Bristol N2). Organelle markers, LETM-1::YFP and wild-type constructs were injected at 1–5 ng/µl. Antisense construct was injected at 50 ng/µl. Feeding RNAi was done by growth according to standard protocols on HT115 bacteria that were transformed with the plL4440 vector with or without the target DNA. The synthesis of dsRNA for injection of adult worms was as described (31). The worms injected with dsRNA were incubated at 20°C and transferred every 12 h. Eggs were counted and survival was scored as growth to the L4 stage. The growth of progeny from feeding RNAi worms was monitored daily by recording their size with photographs. Rescue of C. elegans N2 strain grown on letm-1 feeding RNAi bacteria was done with transgenic animals that express C. briggsae letm-1 genomic DNA fused to GFP under control of the letm-1 promoter (Pletm-1::letm-1::GFP).

Expression of human Letm1
Image clone 4860194, which contains full length human Letm1 cDNA, was obtained from Invitrogen (Carlsbad, CA). This cDNA was recloned into the mammalian expression vector pCDNA3 (Invitrogen, Carlsbad, CA) and, where indicated, adding a GFP or a 3 x HA epitope tag to the C-terminus by PCR. The clones were sequenced to rule out errors introduced by PCR. For bacterial expression, the middle and C-terminal domains of Letm1 were PCR amplified and cloned into pet21d (Novagen Inc., Madison, WI), which adds a 6-his tag for protein purification. Bacterial expression and protein purification was done with standard protocols.

Antibodies
Two different polyclonal antibodies were raised against a bacterially expressed fragment of Letm1, spanning residues 230–482 or 517–739. Antibodies were made in rabbits by Robert Sargeant (Ramona, CA). These antibodies were purified with western blotted recombinant protein as described (32). Glutamate dehydrogenase antibodies were purchased from Rockland (Gilbertsville, PA), TIM23 antibodies from BD Biosciences (Lexington, KY), Porin antibody from Calbiochem (San Diego, CA), ATP synthase {alpha} subunit antibody from Molecular Probes (Eugene, OR), Tubulin antibody from Sigma (St Louis, MO) and HA antibodies from Roche (Indianapolis, IN). GFP antibody was a gift from Dr Greg Payne (UCLA).

Mammalian cell culture and transfection
Wolf–Hirschhorn patient fibroblasts GM00072, GM00343 and GM04126 were obtained from the Corriell Cell Repositories (Camden, NJ). These cells were grown in MEM BSS 2 x concentration of essential and minimum essential amino acids with 2 ml of L-glutamine. HeLa cells and 293T cells were grown in DMEM with 10% fetal calf serum. COS7 cells were grown in DMEM with 5% fetal calf serum. Plasmid DNA was transfected using FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN) and the protocol provided by the manufacturer. Letm1 expression was knocked down by transfecting cells with siRNA oligonucleotides as described (33). Synthetic oligonucleotides, matching the sequence from positions 835–857, were made by Proligo (Boulder, CO). A control duplex was designed by inverting this sequence. The siRNA duplexes were transfected at a concentration of 20 nM into HeLa cells grown to 40% confluency using OligofectAMINETM (Invitrogen, Carlsbad, CA) as described by the manufacturer. Cells were plated in glass-bottom dishes (Mat-Tek, Ashland, MA) and after 3 days they were stained with MitoTracker for fluorescence microscopy. Cells that were intended for electron microscopy were plated on Thermanox® plastic coverslips (Nalge Nunc, Napervilee, IL).

Immunofluorescence and electron microscopy
Cells grown on glass coverslips were fixed for 30 min at room temperature by incubating with pre-warmed 3.7% formaldehyde followed by permeabilization for 10 min at room temperature with 0.1% Triton X-100. Cells were incubated overnight with primary antibody, followed by a 20 min incubation with secondary antibody. Where indicated, cells were stained for 10 min with 0.1 µM MitoTracker Red (Molecular Probes, Eugene, OR) prior to fixation. Images were acquired with a 100 x Neofluar/NA1.3 objective on a Zeiss Axiovert 200 M microscope, using a Hamamatsu ORCA ER camera controlled by Zeiss Axiovision software. In preparation for electron microscopy, cells were fixed for 30 min in 1% glutaraldehyde (Ted Pella Inc., Redding, CA), washed with PBS and incubated for 1 h with 1% osmium tetroxide. The samples were then dehydrated and embedded in Epon resin. Seventy-nanometer-thick sections were stained with uranyl acetate and lead citrate. The sections were viewed with a JEOL-electron microscope (JEOL Ltd, Tokyo, Japan).

Subcellular fractionation and western blotting
Fractionation procedures were as previously described (34). Protease protection experiments were done with mitochondria bovine brain, while carbonate extractions and differential centrifugation were done with mitochondria isolated from HEK 293T cells. Briefly, 50 g fresh bovine brain or HEK 293T cells harvested from nine confluent 150 cm2 plates were homogenized in isolation buffer (70 mM sucrose, 220 mM mannitol, 2 mM HEPES, 0.5 mg/ml BSA) with protease inhibitor mix (Roche Diagnostics GmbH, Mannheim, Germany) with three passes with a loose-fitting pestle in a Potter–Elvejhem homogenizer, followed by five passes with a tight-fitting pestle. The homogenate was subjected to differential centrifugation as described previously (34). The P2 fractions used for protease sensitivity experiments were washed in isolation buffer without protease inhibitors.

Protease protection experiments were done by resuspending 0.5 mg of mitochondrial proteins in 0.8 ml isolation buffer. Trypsin and digitonin were added at the indicated concentrations. After 30 min incubation on ice, the reaction was stopped by adding trichloroacetic acid. Upon precipitation of the proteins, the pellets were resuspended in Laemmli sample buffer and size fractionated by SDS–PAGE. Alkaline extraction was performed by incubating the mitochondrial fraction for 30 min on ice with 0.2 M Na2CO3 adjusted to pH 13 with 1 N NaOH. The membranes were then pelleted by centrifugation for 1 h at 100 000 g. The pellets were resuspended in Laemmli sample buffer, the pH was readjusted and the samples were size fractionated by SDS–PAGE. Proteins that were size-fractionated by SDS–PAGE were transferred to nitrocellulose for western blotting. These blots were incubated with primary antibodies and developed with a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ).

For coprecipitation, HEK 293T cells were transfected with constructs expressing Letm1 fused to three HA tags and Letm1 fused to GFP. The transfected cells were grown for 167emsp14;h, the culture medium was changed and cells were grown for another 8 h, followed by harvesting and lysis with 150 mM NaCl, 5 mM EDTA, 1 mM PMSF, 50 mM Tris–HCl, pH 7.5, 0.4% Triton-X100 and protease inhibitor cocktail (Roche Biochemicals, Indianapolis, IN). Cell debris was removed by centrifugation for 5 min at 10 000g. The supernatant was incubated with antibody for 16 h on ice. The immune complex was incubated with protein G agarose beads (Roche Biochemicals, Indianapolis, IN), pelleted and eluted from the beads by boiling in 1 x Laemmli sample buffer. As controls, lyates were prepared from cells transfected with either construct separately. Immunoprecipitation was then performed on the individual lysates and on a mixture of the two.

Column chromatography
This protocol was adapted from one used previously to determine the size of the fzo1 complex in yeast (35). Mitochondria were isolated as described above and resupended at a concentration of 5 mg protein/ml in lysis buffer (0.4% Triton X-100, 150 mM NaCl, 5 mM EDTA, 1 mM PMSF and 10 mM Tris–Cl pH 7.5). After 30 min at 4°C under constant rocking, the remaining insoluble fraction was pelleted by centrifugation for 20 min at 287 000g. The supernatant was loaded on a 25 ml Superose-12 gel filtration column (Amersham Pharmacia Biotech). The flow rate for chromatography was 0.5 ml/min. Fractions of 0.5 ml were collected and analyzed by SDS–PAGE followed by western blotting and probing with Letm1 antibodies. Size standards were thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa) and aldolase (158 kDa), which were purchased from GE Healthcare (Dallas, TX).


   ACKNOWLEDGEMENTS
 
We thank the other lab members for helpful discussions. We thank Drs L. Griparic and S. Gandre from our lab for some of the samples used for biochemical characterization. We thank the Caenorhabditis Genetics Center (University of Minnesota, St Paul, MN) and by Dr S. Mitani of Tokyo Women's Medical University School of Medicine and the National Bioresource project of Japan for providing C. elegans strains. This work was supported by grants from the American Cancer Society (RSG-01-147-01-CSM) and the National Institutes of Health (GM051866).

Conflict of Interest statement. None declared.


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S. Tamai, H. Iida, S. Yokota, T. Sayano, S. Kiguchiya, N. Ishihara, J.-I. Hayashi, K. Mihara, and T. Oka
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