Examination of Bioenergetic Function in the Inner Mitochondrial Membrane Peptidase 2-Like (Immp2l) Mutant Mice
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Redox Biology 2 (2014) 1008–1015 Contents lists available at ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox Research Paper Examination of bioenergetic function in the inner mitochondrial membrane peptidase 2-like (Immp2l) mutant mice Manish S. Bharadwaj a, Yu Zhou b, Anthony J. Molina a, Tracy Criswell b, Baisong Lu b,n a Section on Gerontology and Geriatric Medicine, Wake Forest University Health Sciences, Department of Internal Medicine, Medical Center Boulevard, Winston-Salem, NC 27157, USA b Wake Forest Institute for Regenerative Medicine, Wake Forest University Health Sciences, Medical Center Boulevard, Winston-Salem, NC 27157, USA article info abstract Article history: Inner mitochondrial membrane peptidase 2-like (IMMP2L) protein is a mitochondrial inner membrane Received 13 August 2014 peptidase that cleaves the signal peptide sequences of cytochrome c1 (CYC1) and mitochondrial glycerol Received in revised form phosphate dehydrogenase (GPD2). Immp2l mutant mice show infertility and early signs of aging. It is 14 August 2014 unclear whether mitochondrial respiratory deficiency underlies this phenotype. Here we show that the Accepted 25 August 2014 intermediate forms of GPD2 and CYC1 have normal expression levels and enzymatic function in Immp2l Available online 28 August 2014 mutants. Mitochondrial respiration is not diminished in isolated mitochondria and cells from mutant Keywords: mice. Our data suggest that respiratory deficiency is not the cause of the observed Immp2l mutant Immp2l phenotypes. Cytochrome c1 & 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND GPD2 license (http://creativecommons.org/licenses/by-nc-nd/3.0/). Complex III Mitochondrial respiration Introduction reductase (Mcr1p) and mitochondrial glycerol-3-phosphate dehy- drogenase (Gut2p) [2–5], whereas Imp2p cleaves the signal Cytochrome c1 (CYC1) and mitochondrial glycerol phosphate sequence from cytochrome c1 [6]. dehydrogenase (GPD2) are mitochondrial inner membrane pro- Mammalian IMMP1L and IMMP2L are homologs of yeast Imp1p teins that need bipartite signal sequences to guide them to their and Imp2p [7,8]. Mouse IMMP2L has two substrates, CYC1 and final destination: the mitochondrial-targeting signal sequence and GPD2 [9]. However, in yeast, Gut2p (a homolog of GPD2) is a the mitochondrial inner membrane-targeting signal sequence. The substrate for Imp1p [5]. Substrates for mammalian IMMP1L have mitochondrial targeting signal sequence guides the precursor not been described, although mammalian DIABLO/SMAC was proteins to the mitochondrial matrix, and is then cleaved by suggested as an IMMP1L substrate [8]. Yeast Imp1p substrate mitochondrial processing peptidase in the matrix. The mitochon- Cyb2p lacks a mammalian ortholog. Yeast Imp1p substrates Cox2p drial inner membrane-targeting signal sequence guides the inter- and Mcr1p have mammalian orthologs, but appear to lack a typical mediate proteins to the inner membrane, and is finally cleaved by cleavable signal peptide sequence [10]. mitochondrial inner membrane peptidase. Yeast mitochondrial Immp2l mutant mice show a series of abnormalities in different inner membrane peptidase has one noncatalytic subunit Som1p organs, which can be classified as either age- dependent or [1], and two catalytic subunits Imp1p and Imp2p, each with non- -independent. Age-independent phenotypes include infertility overlapping substrates. Imp1p cleaves the mitochondrial inner and reduced food intake, which are proposed to be caused by membrane-targeting signal sequences from cytochrome c oxidase superoxide negation of nitric oxide [9,11]. Age-dependent pheno- subunit 2 (Cox2p), cytochrome b2 (Cyb2p), NADH-cytochrome b5 types include spermatogenic damage in male mice [12], bladder dysfunction [13], and ataxia in old mutant mice but not in age- matched normal control mice [14]. These phenotypes are pro- Abbreviations: IMMP2L, inner mitochondrial membrane peptidase 2-like; CS, posed to be caused by chronic oxidative stress resulting from citrate synthase; CYC1, cytochrome c1; ECAR, extracellular acidification rate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase (GAPDH); GPD2, mitochon- increased mitochondrial superoxide generation. While the oxida- drial glycerol phosphate dehydrogenase; OCR, oxygen consumption rate; VDAC1, tive stress hypothesis is consistent with the observed phenotypes, voltage-dependent anion channel 1; complex III, ubiquinol-cytochrome-c reduc- it remains unclear whether the phenotypes are caused by func- tase; complex I:NADH, ubiquinone oxidoreductase; complex II, succinate-ubiqui- tional deficiency of IMMP2L substrates. none oxidoreductase n Corresponding author. CYC1 is one of the 11 subunits of complex III (ubiquinol- E-mail address: [email protected] (B. Lu). cytochrome-c reductase) [15], which is located on the http://dx.doi.org/10.1016/j.redox.2014.08.006 2213-2317/& 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/). M.S. Bharadwaj et al. / Redox Biology 2 (2014) 1008–1015 1009 mitochondrial inner membrane involved in mitochondrial electron Preparation of mitochondrial and cytosolic proteins from mouse transfer. Ubiquinol from complex I (NADH:ubiquinone oxidore- tissues ductase) and complex II are oxidized by mitochondrial complex III to reduce cytochrome c, where CYC1 is the subunit transferring Testicular and brown fat tissues were excised from 2 to the electron to cytochrome c [16]. In yeast, the intermediate form 3-month-old mice. Mitochondrial and cytosolic proteins were of Imp2p substrate Cyt1p (i-Cyt1p, with the inner membrane prepared as described previously [20]. Protease inhibitors targeting signal sequence uncleaved) has normal localization in (0.5 mM PMSF and 1 Â Complete Protease Inhibitor Cocktail from the mitochondria and is respiratory competent [6]. However, the Roche) were included to prevent protein degradation. N-terminal signal peptide sequences of yeast Cyt1p and its mouse homolog CYC1 show little homology, and the entire mature SDS-PAGE and Western blot analyses sequences are only 58% identical. In addition, yeast and mouse complex III have different numbers of subunits [17]. Furthermore, Tissue extracts were prepared from 2 to 3-month-old mice in patients with CYC1 missense point mutations showed reduced RIPA buffer including protease inhibitors. Mitochondrial, cytosolic complex III activity and insulin-responsive hyperglycemia [18]. proteins, and tissue extracts were separated by SDS-PAGE, trans- Thus, it remains unclear whether the enzymatic function of the ferred to a nitrocellulose membrane, and incubated with anti β- intermediate form of CYC1 (i-CYC1) is affected in Immp2l mutant actin (Sigma, 1:5000), anti-GAPDH (Abcam, 1:500), anti-CYC1 mice. (ProteinTech Group, 1:1000), anti-GPD2 (Abnova, 1:500), anti- GPD2, another IMMP2L substrate, catalyzes the conversion of VDAC1 (Abcam, 1:2000). Horseradish peroxidase (HRP)-conju- glycerol-3-phosphate to dihydroxyacetone phosphate, reducing gated secondary antibodies were purchased from Pierce. Chemi- the enzyme-bound FAD. Together with the cytosolic glycerol luminescent reagents from Pierce were used to visualize the fi phosphate dehydrogenase (GPD1), they form the glycerol phos- protein signals under the LAS-3000 system from Fuji lm. The phate shuttle, which uses the interconversion of glycerol-3-phos- Integrated Density function from ImageJ software was used to quantify the expression of individual proteins after normalization phate and dihydroxyacetone phosphate to transfer reducing β equivalents into mitochondria, resulting in the reoxidation of by -actin or GAPDH (for cytosolic proteins or total tissue extracts) NADH formed during glycolysis. It also remains unclear whether and VDAC1 (for mitochondrial proteins). the enzymatic activity of GPD2 is impaired in Immp2l mutant mice due to the presence of uncleaved signal sequences. Oxygen consumption rate (OCR) of isolated mitochondria The present study was designed to address questions regarding the enzymatic activities of IMMP2L substrates in Immp2l mutant Whole hind limb skeletal muscle from a control and an À/À – mice. We find that CYC1 and GPD2 are functional in Immp2l Immp2l mouse (2 3 months) was used for mitochondria – mutant mice and conclude that mitochondrial respiration defi- isolation, following the protocols described previously [21 23]. Mitochondrial protein concentrations were determined using the ciency is not the cause of the observed phenotypes. BCA protein assay kit (Thermo Scientific, Rockford, IL). Mitochon- drial OCR was measured using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse Biosciences, Billerica, MA). 10 mM succinate Materials and methods was used as the substrate with the addition of 2 mM rotenone; 5 mg mitochondrial protein was loaded in each well. Three to four Animals replicates of each sample were analyzed for the mean and SD. Following an initial 7 min of equilibration, ADP (2 mM), oligomy- The development and characterization of the Immp2l mutant cin (2 mM), FCCP [carbonyl cyanide 4-(trifluoromethoxy) phenyl- mice has been described previously [9]. Mice were housed in the hydrazone, 6 mM] and antimycin (2 mM) were added successively pathogen-free animal facility of Wake Forest University Health to achieve the final concentration as given in parentheses. Sciences. Experiments were conducted in accordance with the National Research Council publication