LACTB Is a Filament-Forming Protein Localized in Mitochondria

LACTB is a filament-forming protein localized in mitochondria

Zydrune Polianskytea, Nina Peitsaroa, Arvydas Dapkunasa, Julius Liobikasa,1, Rabah Soliymanib, Maciej Lalowskib, Oliver Speera,2, Jani Seitsonenc, Sarah Butcherc, Grazia M. Cereghettid, Matts D. Lindere, Michael Merckela, James Thompsonf,g, and Ove Erikssona,3

aResearch Program of Molecular Neurology, Institute of Biomedicine, bProtein Chemistry Unit, Biomedicum Helsinki, cInstitute of Biotechnology, Department of Biological and Environmental Sciences, and eDepartment of Anatomy, Institute of Biomedicine, University of Helsinki, FIN-00290 Helsinki, Finland; dDepartment of Cell Physiology and Metabolism, University of Geneva, CH-1221 Geneva, Switzerland; fHigh Throughput Center, Department for Biosciences and Medical Nutrition, Karolinska Institute, S-141 57 Huddinge, Sweden; and gMolecular Medicine Techology Center, Institute for Molecular Medicine Finland (FIMM), Biomedium, Helsinki 2, Finland

Edited by Peter A. Reichard, Karolinska Institutet, Stockholm, Sweden, and approved September 10, 2009 (received for review June 18, 2009) LACTB is a mammalian active-site serine protein that has evolved expression analysis based on data integrated from multiple from a bacterial penicillin-binding protein. Penicillin-binding pro- sources (9). This finding was subsequently validated in vivo teins are involved in the metabolism of peptidoglycan, the major through LACTB overexpression in transgenic mice, which re- bacterial cell wall constituent, implying that LACTB has been sulted in an obese phenotype (9). Although the biochemical endowed with novel biochemical properties during eukaryote mechanism for the obesity-promoting effect of LACTB remains evolution. Here we demonstrate that LACTB is localized in the unclear, it is evident that LACTB can affect whole-organism mitochondrial intermembrane space, where it is polymerized into energy homeostasis and therefore that LACTB is directly or stable ﬁlaments with a length extending more than a hundred indirectly involved in the regulation of the metabolic circuitry. nanometers. We infer that LACTB, through polymerization, pro- In this study we have performed a molecular dissection to motes intramitochondrial membrane organization and micro-com- elucidate the biochemical function of LACTB. We show that partmentalization. These ﬁndings have implications for our under- LACTB, unlike any known bacterial PBP family protein, can

standing of mitochondrial evolution and function. polymerize into stable filaments occupying the mitochondrial BIOCHEMISTRY intermembrane space. We speculate that LACTB filaments may itochondria descend from ancient Gram-negative bacteria play a role in submitochondrial organization and therefore Mthat, through endosymbiosis, became permanent residents possibly affect mitochondrial metabolon organization. of eukaryotic cells (1–3). As a consequence, mitochondria and Gram-negative bacteria share several biochemical features, in- Results and Discussion cluding DNA organization, core metabolism, and a double- LACTB Is Localized in the Mitochondrial Intermembrane Space. membrane architecture. In Gram-negative bacteria, but not in LACTB is widely expressed in different mammalian tissues (7, mitochondria, a mesh-like layer of peptidoglycan is deposited 10–13). Although proteome survey studies show that LACTB is between the outer and inner membrane, offering protection associated with mitochondria (10–13), other studies suggest that against mechanical stress. Following endosymbiosis, the pepti- LACTB is localized in non-mitochondrial compartments (14, doglycan layer lost its structural importance, and was subse- 15). Therefore, we used a set of complementary experimental quently eliminated from the early eukaryotic cell. Although techniques to determine the subcellular localization of LACTB. eukaryotes lack peptidoglycan, proteins deriving from the pen- First, we used HeLa cells genetically modified to express red icillin-binding protein (PBP) family (4) are found in all major fluorescent protein (RFP) exclusively in mitochondria (mtRFP). eukaryotic lineages, including vertebrates (5). Endogenous LACTB of mtRFP HeLa cells was detected with an The bacterial PBPs constitute a large family of serine pro- anti-LACTB antibody (Fig. 1A). LACTB and mtRFP displayed teases that is distinguished by 3 conserved amino acid motifs that a similar intracellular distribution, confirming that LACTB is contribute to the formation of the catalytic site. The -SXXK- associated with mitochondria. The N-terminal 97 aa segment of motif contains the catalytic serine residue, which undergoes LACTB does not form part of the conserved PBP domain and reversible acylation through substrate binding, whereas the may therefore be responsible for organelle targeting (5, 7). To -[SY]X[NT]- and the -[KH][ST]G- motifs contribute to substrate investigate if the N-terminal amino acid segment of LACTB docking (4). PBPs catalyze the hydrolysis or transpeptidation of influences its subcellular localization, we fused GFP to the the terminal D-alanyl-D-alanine moiety in peptidoglycan stem C-terminus of WT LACTB and also to a truncated LACTB peptides. The acceptor for the transpeptidation reaction is the lacking the first 97 aa. Expression of these fusion proteins in ␧-amino group of lysine or diaminopimelate in an adjacent stem mtRFP HeLa cells confirmed that the N-terminal segment of peptide. Through these reactions, PBPs contribute to modulate the extent of peptidoglycan cross-linking during bacterial cell Author contributions: Z.P., N.P., J.L., O.S., S.B., and O.E. designed research; Z.P., N.P., A.D., division and cell wall elongation (4). J.L., R.S., O.S., M.D.L., and O.E. performed research; G.M.C. and M.M. contributed new In contrast, the function of PBP homologues in eukaryotic reagents/analytic tools; Z.P., N.P., A.D., J.L., R.S., M.L., O.S., J.S., S.B., M.D.L., J.T., and O.E. organisms remains largely unexplored. Amino acid sequence analyzed data; and Z.P., N.P., J.T., and O.E. wrote the paper. analyses show that the 3 conserved amino acid motifs required The authors declare no conﬂicts of interest. for catalytic activity are conserved in all eukaryotic PBP homo- This article is a PNAS Direct Submission. logues (5), suggesting that they can function as active-site serine Freely available online through the PNAS open access option. enzymes. Within the metazoan division, nematodes harbor the 1Present address: Laboratory of Biochemistry, Institute for Biomedical Research of Kaunas largest number of PBP homologues (5), and in Caenorhabditis University of Medicine, LT-50009 Kaunas, Lithuania. elegans the PBP homologue LACT-1 may be involved in patho- 2Present address: Division of Hematology, University Children’s Hospital Zurich, CH-8032 gen recognition (6). LACTB is the only PBP homologue of Zu¨rich, Switzerland. mammals, and LACTB has been identified in all mammalian 3To whom correspondence should be addressed. E-mail: ove.eriksson@helsinki.ﬁ. genomes sequenced to date (5, 7, 8). Recently, a causative link This article contains supporting information online at www.pnas.org/cgi/content/full/ between LACTB and obesity was detected through gene co- 0906734106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906734106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 1, 2021 Fig. 1. Subcellular localization of LACTB in HeLa cells and rat tissues. (A and B) Fluorescence microscopy images of mtRFP. (A) Endogenous LACTB visualized with an anti-LACTB antibody and an Alexa 488-coupled secondary antibody. The region marked with a square is enlarged (Inset). (B) Transfection with plasmid constructs encoding WT LACTB or an N-terminally truncated LACTB fused to a C-terminal GFP (wt-LACTB-GFP, and ⌬1–97-LACTB-GFP). (C) Immunoblotting of proteins from rat liver homogenate (H), cytosolic fractions (S1-S3), and mitochondrial fraction (Mito). (D) Immunoblotting of LACTB from rat liver mitochondria incubated with digitonin and trypsin compared with compartment-speciﬁc markers for intermembrane space (IMS), matrix, and outer membrane (OM). (E) Soluble mitochondrial proteins were separated from integral membrane proteins through extraction with Na2CO3. The membrane fraction (Mf) and soluble fraction (Sf) were applied on SDS/PAGE gels and analyzed by immunoblotting. (F) Rat tissue cryo-sections immuno-labeled with an anti-LACTB antibody displayed by nanogold particles. The particle densities were 5.9 ␮mϪ2 over mitochondria and 0.8 ␮mϪ2 elsewhere (n ϭ 3,820). Particles located over cristae are marked with green arrows.

LACTB is necessary for mitochondrial localization (Fig. 1B). (Opa-1) (17), became accessible to trypsin only upon outer These results indicate that WT LACTB resides exclusively inside membrane solubilization (Fig. 1D). To determine if LACTB is a mitochondria. soluble protein or an integral membrane protein, mitochondria Next, we prepared tissue fractions from rat livers. Proteins were treated with sodium carbonate followed by centrifugation extracted from the different tissue fractions were analyzed by to precipitate the membranes. Immunoblotting of the resulting immunoblotting using antibodies against LACTB and compart- fractions showed that LACTB was completely separated from ment-specific marker proteins. In crude rat liver extracts, the the membrane marker proteins porin and prohibitin, indicating anti-LACTB antibody revealed a single 55 kDa band, which, as that LACTB is a soluble protein (Fig. 1E). Last, we prepared predicted, had segregated with the mitochondrial fraction (Fig. samples of cryo-sectioned rat tissues for immuno-electron mi- 1C). To determine in which intra-mitochondrial compartment croscopy. LACTB was visualized by nanogold particles coupled LACTB resides, we incubated the mitochondria with trypsin and to the anti-LACTB antibody. The results revealed individual sufficient digitonin to permeabilize the outer, but not the inner, nanogold particles, or, less frequently, large particle clusters over mitochondrial membrane. Immunoblotting revealed that the intermembrane space of mitochondria (Fig. 1F). We con- LACTB, like other intermembrane space proteins such as apo- clude that LACTB resides in the mitochondrial intermembrane ptosis-inducing factor (AIF) (16) and optic atrophy protein 1 space.

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0906734106 Polianskyte et al. Downloaded by guest on October 1, 2021 LACTB Forms a Soluble High Molecular Weight Homopolymer. The molecular machineries for several essential metabolic processes are localized in the mitochondrial intermembrane space. To investigate if LACTB is associated with any of these processes, we extracted intermembrane space proteins using non- denaturing conditions. Proteins were separated by 2D blue native SDS/PAGE for identification by immunoblotting and MS. Immunoblotting with the anti-LACTB antibody revealed a band ranging from 600 kDa to several MDa in the native direction of the gel (Fig. 2A). However, in the denaturing direction of the gel, this band separated into a major 55 kDa component and a minor 200 kDa component [Fig. 2A and supporting information (SI) Fig. S1]. This finding suggests that LACTB is part of a multi- protein complex or exists as a homopolymer. Analyses of the major immunoreactive band by MS yielded high-scoring MS/MS spectra from peptides covering almost half of LACTB’s amino acid sequence (Fig. 2 B-D and Fig. S2). Notably, we obtained an MS/MS spectrum of a 3,676.3 Da peptide that could be assigned to a 38 aa segment starting from alanine 63 (Fig. 2C). However, the amino acid sequence of the N-terminal cleavage site of this peptide shows that it could not have been formed by the trypsin used for sample preparation. This implies that the 3,676.3 Da peptide is located at LACTB’s N-terminus and indicates that mature LACTB is formed from the preprotein by removal of 62 aa (Fig. 2B). This cleavage site is preceded by a segment of hydrophobic amino acids (Fig. 2B), suggesting that LACTB is imported to the intermembrane space through a bipartite pre-

sequence cleaved first by the mitochondrial processing peptidase BIOCHEMISTRY followed by a second cleavage by a protease at the outer surface of the inner membrane (19). Having defined the mature 55 kDa LACTB protein, we proceeded with the MS analysis of the remaining region (indicated in Fig. 2A) of the 2D blue native SDS gel. This analysis resulted in the identification of LACTB in the minor 200 kDa immunoreactive band as well; however, it did not yield any protein that could be a possible LACTB binding partner (Table S1). Therefore, these findings support the hy- pothesis that LACTB is forming a homopolymer.

LACTB Is Polymerized into Ordered Filaments. To investigate the structure of the high molecular mass form of LACTB, we used EM. Extracted intermembrane space proteins were separated by centrifugation in a CsCl-density gradient and the resulting fractions were examined after negative staining. At a gradient density of 1.25 to 1.28 g/cm3, we observed characteristic filaments of various lengths composed of globular subunits (Fig. 3A). We noted that, as a result of their heterogeneous size, these filaments would probably migrate over a broad molecular mass range upon electrophoretic separation, suggesting that they represented LACTB polymers. Subsequent MS analyses confirmed that the 1.25 to 1.28 g/cm3 CsCl-gradient fraction contained LACTB. Using whole-mount immuno-electron microscopy, we showed that the anti-LACTB antibody labeled the filaments (Fig. 3B). These findings demonstrate that LACTB can polymerize and form filaments with an open symmetry. To Fig. 2. Analysis of LACTB by MS. (A) Soluble mitochondrial intermembrane investigate the organization of the LACTB polymer, we aligned space proteins separated by 2D blue native SDS/PAGE followed by immuno- and averaged images of 128 subunits (Fig. S3). Using tilting and blotting with an anti-LACTB antibody. The region boxed by dashed lines was averaging, the subunit volume was calculated to be 360 nm3. This analyzed by MS. (B) Amino acid sequence of rat LACTB (RefSeq:XP࿝217181). volume could, depending on packing constraints, accommodate Peptides identiﬁed by MS/MS are highlighted. The deduced cleavage site for 3 to 5 molecules of LACTB, implying a subunit mass of 150 to the mitochondrial import pre-sequence, marked with an arrow, displays a tetrapeptide motif (AVPI-) shared by several mitochondrial intermembrane 250 kDa. This size is in agreement with the molecular mass of 200 space proteins (18). The hydrophobic amino acid segment located adjacent to kDa observed for the minor LACTB band in the 2D gels (Fig. the cleavage site is marked with a red line. (C and D) MS/MS spectra, amino acid 2A and Fig. S1), suggesting that the LACTB polymer is com- sequence, and fragment ions from peptides assigned to the N-terminal (C) and posed of tetrameric subunits. the C-terminal (D) segments of LACTB.

Stable LACTB Filaments Are Formed Inside Mitochondria. To investigate if LACTB polymers occur in situ, we prepared mitochon- formed clusters in the intermembrane space (Fig. S4), this dria for immuno-electron microscopy. Although cryo-sections approach yielded no further ultrastructural information. In labeled with the anti-LACTB antibody confirmed that LACTB contrast, chemical fixation of mitochondria under conditions

Polianskyte et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on October 1, 2021 nents. Importantly, these findings point to the possibility that LACTB has a structural function. We estimated the amount of LACTB in mitochondria by using quantitative immunoblotting. We found that a liver mitochon- drion contained, on average, 1,500 molecules of LACTB (Fig. S6 and Table S2 for a comparison with other mitochondrial proteins). We conclude that LACTB is an abundant mitochondrial protein and therefore has the potential to impact mitochondrial ultrastructure.

Polymerization Is Probably a Unique Feature of LACTB and Its Meta- zoan Orthologues. To gain insight into the molecular basis for LACTB’s polymerization, we analyzed the amino acid sequence for secondary structure motifs predicted to mediate protein- protein interactions. The analysis revealed a segment enriched in charged and hydrophobic amino acids showing a high propensity for coiled-coil motif formation (Fig. 4 A and B). To assess if this region could be positioned for protein-protein interactions, we performed homology modeling using the crystal structure of the Streptomyces R61 D-alanyl-D-alanine carboxypeptidase (20), an extensively studied bacterial homologue of LACTB. According to our model, LACTB exhibits a characteristic PBP fold con- sisting of an ␣/␤ region and an all-helical region (Fig. 4C). The predicted coiled-coil segment is positioned on LACTB’s surface, permitting formation of a flexible loop (Fig. 4C). The loop may Fig. 3. Visualization of LACTB by transmission EM. (A and B) Proteins from CsCl-gradient fractions visualized through negative staining with uranyl ace- contribute to the formation of complementary patches, thereby tate. (C and D) Thin sections of mitochondria and submitochondrial particles promoting self-assembly of the LACTB polymer. after chemical fixation, embedding, and staining with uranyl acetate and lead citrate. (A) Naked filaments and (B) filaments decorated with anti-LACTB Concluding Remarks. Proteins forming polymers with an open antibodies and nanogold particles (red arrows). The particle densities were symmetry typically have structural functions that are conserved 540 ␮mϪ2 over filaments and 3 ␮mϪ2 elsewhere (n ϭ 148). (C) Isolated rat liver over long evolutionary distances. This is exemplified by the 3 mitochondria with filaments in the cristal part of the intermembrane space main cytoskeletal elements of eukaryotic cells; actin, tubulin, the (green arrows). (D) SMPs containing entrapped filaments (green arrows; see intermediate filament proteins, and their respective bacterial also Fig. S5). Previous investigators have described intramitochondrial filaments in various tissues from several different species, including humans. homologues; MreB, FtsZ, and crescentin. In contrast, PBPs are Reports on filaments that, based on their location and geometry, may repre- not known to assemble into filaments but instead promote sent LACTB are listed in Table S3. subcellular structure formation through enzymatic mechanisms. The difference between a possible structural function of LACTB exerted through filament formation and the overtly enzymatic optimized for ultrastructural preservation revealed organized function of PBPs is very large. This raises intriguing questions as filaments (Fig. 3C and Movie S1) occupying distinct regions of to what advantage intramitochondrial filaments conferred to the the intermembrane space. Like polymeric LACTB, these fila- eukaryotic cell and why LACTB was endowed with the property ments were composed of globular subunits arranged in straight to self-assemble into filaments. One may hypothesize that, after or slightly curved strands. Measurements showed that these the loss of the peptidoglycan layer, novel molecular mechanisms filaments and LACTB polymers shared a similar thickness [13.7 for mitochondrial intermembrane space organization had to be nm Ϯ 0.2 vs. 15.5 nm Ϯ 0.1 (SEM), n ϭ 25] and repeat distance developed, and that gene regulatory mechanisms favored evo- [15.4 nm Ϯ 0.1 vs. 14.4 nm Ϯ 0.1 (SEM), n ϭ 150]. These findings lutionary tinkering of proteins previously functioning in that demonstrate that LACTB polymers can form in situ, suggesting context. that the polymerization is an ordered process with a specific Recent results from gene network analyses and a transgenic physiological function. mouse model indicate that LACTB can affect the energy balance We noted that LACTB filaments were confined to the intra- leading to an increased fat storage (9). We speculate that cristal region of the intermembrane space, i.e., to the region LACTB has a physiological role in promoting submitochondrial formed by infoldings of the inner membrane, and that the organization, thereby affecting metabolite flux through specific loci in the metabolic web. filament ends appeared to be tethered to the inner membrane (Fig. 3C and Movie S1). The uniform organization of the Experimental Procedures LACTB filaments relative to the inner membrane suggested that LACTB Expression Plasmids. Plasmids for LACTB expression were generated they interact in a specific way. To investigate this issue, we used using the Gateway cloning system (Invitrogen), as previously described (8). submitochondrial inner membrane vesicles (SMPs). During SMP preparation, mitochondria are disrupted and freely diffusing Cell Culture. HeLa cells were stably transfected with a plasmid encoding for a molecules are lost. As a consequence, SMPs contain only mitochondria-targeted RFP (mtRFP). HeLa cells were transiently transfected membrane components and membrane-bound macromolecules with LACTB constructs cloned into the pcDNA-DEST47 plasmid (Invitrogen) (Fig. S5). Chemical fixation of SMPs followed by EM analysis using the FuGENE HD Transfection Reagent (Roche) according to the manu- revealed that they harbored intact LACTB filaments (Fig. 3D). facturer’s instructions. Cells were fixed with 4% paraformaldehyde, washed, and embedded for fluorescence microscopy. The filaments spanned across the entire vesicle interior and were tethered to the vesicle membrane at the ends. We conclude that Isolated Mitochondria. Mitochondria were isolated from livers of male Wistar LACTB filaments are spatially organized structures, forming a rats, as previously described (21). Proteins were extracted from the mitochon- stable module via interactions with inner membrane compo- drial intermembrane space by using a hypo-osmolar medium to break the

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Fig. 4. Structural model of LACTB. (A) Amino acid alignment of predicted coiled-coil segment in metazoan LACTB orthologues and the corresponding regions from 3 bacterial penicillin-binding proteins (protein data bank accession numbers are listed in ref. 5). (B) Amino acid similarity scores for LACTB and Streptomyces R61 D-alanyl-D-alanine carboxypeptidase (colored band) and probability of coiled-coil formation for LACTB (trace). The 3 catalytic site signature motifs -SISK-, -YST-, and -HTG-, universally conserved in penicillin-binding proteins (4), are indicated. (C) Three-dimensional model of LACTB shows the position of the predicted coiled-coil segment (yellow arrows), and the side chains of the catalytic site residues (yellow).

mitochondrial outer membrane. Extracted mitochondrial intermembrane Antibodies. Anti-LACTB antibody was raised, purified, and characterized as space proteins were separated on a CsCl-gradient and then prepared for previously described (8). Anti-actin antibody was from MP Biomedicals, anti- electron microscopy as detailed (22), with minor modifications. SMPs were AIF antibody and anti-cytochrome c antibody were from Millipore, anti-Hsp60 prepared using sonication. antibody and anti-prohibitin antibody were from Santa Cruz Biotechnology, anti-Opa-1 antibody was from BD Biosciences, anti-porin/VDAC antibody was Gel Electrophoresis. One-dimensional SDS/PAGE and 2D blue native SDS/PAGE from Calbiochem, peroxidase-conjugated anti-rabbit IgG antibody and per- were performed using the BioRad MiniPROTEAN and PROTEAN II systems. 2D oxidase conjugated anti-mouse IgG antibody were from Sigma, and Alexa blue native PAGE was performed as detailed (23), with minor modifications. 488-conjugated anti-rabbit antibody was from Invitrogen. Complete details about the experimental procedures can be found in the SI MS. Proteins separated by gel electrophoresis were prepared for MS as pre- Text. viously described (8). MALDI-TOF analyses were performed using a Bruker Autoflex III mass spectrometer. Liquid chromatography-MS analyses were ACKNOWLEDGMENTS. We thank E. Jokitalo and P. Laurinma¨ki for help with electron microscopy at the Institute of Biotechnology; and P. Bernardi for his performed using an HPLC system from LC Packings connected to a Bruker advice and encouragement. This study was funded by grants from the Acad- Esquire 3000 plus ion trap mass spectrometer. emy of Finland, the University of Helsinki Research Foundation, the Sigrid Juselius Foundation, the Finska La¨karesa¨llskapet, the Magnus Ehrnrooth EM. Samples in negative staining were viewed using a Philips Tecnai F20 foundation, the K. Albin Johansson foundation, and the Svenska Kul- electron microscope. Embedded and thin-sectioned samples were viewed turfonden. J.S. was supported by the National Graduate School in Informa- with a JEOL 1200 EX II electron microscope. tional and Structural Biology.

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