Phosphorylation and cleavage of presenilin-associated rhomboid-like protein (PARL) promotes changes in mitochondrial morphology

Danny V. Jeyaraju*, Liqun Xu†, Marie-Claude Letellier*, Sirisha Bandaru*, Rodolfo Zunino†, Eric A. Berg‡, Heidi M. McBride†§, and Luca Pellegrini*§

*Centre de Recherche Universite´Laval Robert-Giffard, 2601 ch. de la Canardie`re, Quebec City, QC, Canada G1J 2G3; †University of Ottawa Heart Institute, 40 Ruskin Street, Ottawa, ON, Canada K1Y 4W7; and ‡21st Century Biochemicals, 33 Locke Drive, Marlboro, MA 01752-1146

Edited by Walter Neupert, Institute fu¨r Physiologische Chemie, Munich, Germany, and accepted by the Editorial Board October 12, 2006 (received for review June 14, 2006) Remodeling of mitochondria is a dynamic process coordinated by residues would be expected to survive the Ϸ100 million years of fusion and fission of the inner and outer membranes of the organelle, evolution separating mammalian orders (12, 13). This analysis mediated by a set of conserved proteins. In metazoans, the molecular suggests that emergence of the P␤ domain at the outset of verte- mechanism behind mitochondrial morphology has been recruited to brate evolution may be associated with the appearance of a new govern novel functions, such as development, calcium signaling, and mechanism of regulation of PARL. We have recently shown that apoptosis, which suggests that novel mechanisms should exist to this part of the PARL molecule undergoes two consecutive cleav- regulate the conserved membrane fusion/fission machinery. Here we age events, termed ␣ and ␤. The proximal ␣-cleavage is a consti- ␤ show that phosphorylation and cleavage of the vertebrate-specific P tutive processing associated with the protein import in the mito- domain of the mammalian presenilin-associated rhomboid-like chondria, whereas the distal ␤-cleavage is regulated through a (PARL) protease can influence mitochondrial morphology. Phosphor- mechanism of proteolysis requiring PARL activity supplied in trans ylation of three residues embedded in this domain, Ser-65, Thr-69, and (11). Whether this cleavage occurs in vivo is unknown. In addition, Ser-70, impair a cleavage at position Ser77–Ala78 that is required to its mechanism of regulation and its functional significance remain initiate PARL-induced mitochondrial fragmentation. Our findings re- unexplored. veal that PARL phosphorylation and cleavage impact mitochondrial dynamics, providing a blueprint to study the molecular evolution of mitochondrial morphology. Results Human PARL Is Subjected to ␤-Cleavage in Vivo. PARL transfected 77 78 protein evolution ͉ protein phosphorylation ͉ rhomboids ͉ mitochondrial in HEK 293 cells is cleaved at position Ser –Ala , which maps dynamics ͉ intramebrane proteolysis within the vertebrate-specific P␤ domain (11), suggesting that, in vivo, the rhomboid protease may undergo the same processing. To itochondrial biogenesis is an essential cellular process address this possibility, we generated a polyclonal antibody against Mgoverned by a small set of proteins with membrane a peptide spanning the C terminus of PARL (anti-PARL-C-Term; pro-fusion and pro-fission activities which are conserved in all Fig. 1A). This specific antibody (Fig. 1B) was used to immunopre- eukaryotes (1–3). During metazoan evolution, this process has cipitate endogenous PARL from lysates of mitochondria isolated been recruited to coordinate novel mitochondrial functions, such from human placenta. Using antibodies recognizing the N-terminal as apoptosis (4–6), thereby suggesting the emergence, in higher and C-terminal regions of PARL (Fig. 1A), we then examined the eukaryotes, of novel mechanisms of regulation of the fusion and cleavage of the endogenous protein relative to the transfected fission machinery of the organelle. Formal, mechanistic evi- PARL-FCT by epitope mapping. Data showed two bands whose dence supporting this hypothesis is, however, still missing. mobility is, as expected, slightly higher than that of the FLAG- Recently, rhomboid proteases have been implicated in the reg- tagged positive control (Fig. 1C Upper). Although both forms are ulation of mitochondrial membrane remodeling. Studies in Sac- immunoreactive against anti-PARL-C-Term, only the slower mi- charomyces cerevisiae demonstrated that PCP1P is required to grating band was positive to anti-PARL-N-Term, indicating that cleave Mgm1p, an intermembrane space dynamin family member the corresponding epitope was absent in the faster migrating band that participates in mitochondrial fusion events (7, 8). The yeast (Fig. 1C). These data strongly suggest that endogenous PARL N PCP1P protein belongs to a subfamily of mitochondrial rhomboid terminus undergoes ␤-cleavage, indicating that this processing may proteases typified by presenilin-associated rhomboid-like (PARL) mechanistically coordinate the function of the rhomboid protease protein (9, 10), the human ortholog of PCP1P (8). Despite their in vivo. functional and structural conservation, PCP1P and PARL have unrelated N-terminal domains. The N-terminal region of PARL shows no detectable similarity to any other available protein Author contributions: D.V.J. and L.X. contributed equally to this work; H.M.M. and L.P. sequences. This region of PARL, designated P␤ (spanning amino designed research; D.V.J., L.X., M.-C.L., S.B., R.Z., E.A.B., H.M.M., and L.P. performed acids 40–100), is vertebrate-specific, as indicated by the notable research; H.M.M. and L.P. contributed new reagents/analytic tools; E.A.B., H.M.M., and L.P. conservation among mammals and, to a lesser extent, other verte- analyzed data; and H.M.M. and L.P. wrote the paper. brates, but not between vertebrates and insects (11). Although the Conflict of interest statement: E.A.B. is an employee and stockholder of Century 21st ␤ Biochemicals. This company sells mass spectrometry analysis and antibody production function of the P domain remains unknown, its biological rele- services. vance is evident from its sequence conservation. Indeed, in the four This article is a PNAS direct submission. W.N. is a guest editor invited by the Editorial Board. available mammalian PARL sequences, 58 of the 62 residues of the ␤ Abbreviations: LC/MS, liquid chromatography/mass spectrometry; MAMP, mature mito- P domain are invariant, and there are no insertions or deletions chondrial PARL; PACT, PARL C-terminal product (of ␤-cleavage); PARL, presenilin-associ- (11), which suggests that at least during mammalian evolution, the ated rhomboid-like (protein); PARL-FCT, PARL-FLAG-CTerminus. N-terminal region of PARL was subject to strong purifying selec- §To whom correspondence may be addressed. E-mail: [email protected] or tion, which can be explained by functional constraints. In uncon- [email protected]. strained sequences evolving neutrally, very few, if any, invariant © 2006 by The National Academy of Sciences of the USA

18562–18567 ͉ PNAS ͉ December 5, 2006 ͉ vol. 103 ͉ no. 49 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604983103 Downloaded by guest on October 2, 2021 obtained from 250 mg of solubilized human placenta mitochondria, it was digested, and the peptides were subjected to LC/MS analysis. Data showed that Ͼ35% of the entire protein was covered (Table 1, which is published as supporting information on the PNAS web site), with two molecular ions spanning nearly the entire P␤ domain of the mitochondrial mature form of PARL, MAMP (Fig. 1A). Ion m/z 1072.932ϩ corresponded to a triple-phosphorylated 60VE- PRRSDPGTSGEAYKR76 peptide, which maps between the ␣- and ␤-cleavage sites; ion m/z 1138.132ϩ corresponded instead to an unmodified peptide spanning the ␤-cleavage site and its distal region (77SALIPPVEETVFYPSPYPIR96;Fig.2A and E). To investigate whether transfected PARL is also phosphorylated, we overexpressed PARL-FCT in HEK 293 cells. The protein was immunoprecipitated with anti-FLAG to isolate the transfected protein. It was unlikely that endogenous PARL was copurified during this step because coimmunoprecipitation studies with PARL constructs harboring different tags did not reveal ho- modimers or oligomers (data not shown). The ␣-cleaved form of PARL, MAMP (Fig. 1A), was then isolated by gel electrophoresis, digested, and subjected to LC/MS analysis. More than 51% of the protein was covered (Table 2, which is published as supporting information on the PNAS web site). Within this peptide data set we also observed a triple-phosphorylated 56APRKVEPRRSDPGTS- GEAYKR76 peptide, molecular ion m/z 866.393ϩ (Fig. 2B), which overlaps with most of the triple- phosphorylated m/z 1072.932ϩ identified during the analysis of the endogenous PARL. Molecular ion m/z 1138.132ϩ, 77SALIPPVEETVFYPSPYPIR96, was also found (Table 2), indicating that sample preparation and analyses were performed under comparable experimental conditions. Sim- ilar results were also obtained from PARL-FCT isolated from transfected HeLa cells (data not shown). To refine these results, we subjected ion m/z 866.393ϩ to tandem MS analysis. Data showed a series of three water and phosphoric acid losses as the primary detected fragments (Fig. 2 D and E and Table 3, which is published as supporting information on the PNAS web site), consistent with the fragmentation pattern of a peptide with phosphorylated Ser and Thr, rather than Tyr residues. Addi- tionally, the nonphosphorylated y3 ion and the Y immonium ion but not their corresponding phosphorylated species were detected, indicating lack of phosphorylation at Tyr-74 of PARL. Further- more, the N-terminal ion b12–H3PO4, an internal ion series (GTSG–2H3PO4, PGTS–2H3PO4, DPGTSG–2H3PO4), and the Fig. 1. Cleavage of PARL P␤ domain in vivo.(A) Schematic representation of C-terminal ion y18–H3PO4 indicate phosphorylation at Ser-65, the ␣- and ␤-cleaved forms of PARL, MAMP, and the PARL C-terminal product Thr-69, and Ser-70 (Fig. 2 D and E and Table 3). This conclusion of ␤-cleavage (PACT). The locations of the epitopes recognized by the anti- was further supported by the lack of phosphorylated peptides in the PARL-N-Term and anti-PARL-C-Term antibodies are indicated. Small black data set obtained from LC/MS analysis of a transfected PARL squares depict the seven transmembrane helixes of PARL. (B) Specificity of the mutant bearing Ala substitutions at these residues (Fig. 2 C and E anti-PARL-C-Term antibody. The anti-PARL-C-Term antibody specifically im- and data not shown). A monophosphorylated peptide spanning munoprecipitates (IP) PARL-FLAG-CTerminus (PARL-FCT) from HEK 293- transfected cells as well as endogenous PARL from mitochondrial lysates of Ser-65, Thr-69, and Ser-70 was detected in vivo as well as in vitro human placenta. The specificity was addressed by preadsorbing the antisera (Table 1); however, its relative abundance was very low compared

with the synthetic peptide PARL-C-Term used to generate the corresponding with the triple- phosphorylated form (data not shown), indicating CELL BIOLOGY antibody. WB, Western blotting. (C) Endogenous PARL is subjected to N- that most of the ␣-cleaved form of PARL is phosphorylated. We terminal ␤-cleavage as observed for transfected PARL. Transfected PARL-FCT conclude that the vertebrate-specific P␤ domain of endogenous and and endogenous PARL were immunoprecipitated with anti-PARL-C-Term and transfected PARL is phosphorylated at residues Ser-65, Thr-69, subjected to epitope mapping. (Upper) Both the transfected and endogenous and Ser-70, and, by implication, that this modification has the same PARL are present in two forms, MAMP and PACT, which migrate according to function in vivo and in vitro. the predicted molecular mass shown in A. The lower band labeled as PACT- FLAG-CT corresponds to the product of ␤-cleavage at residue Ser77–Arg78 (11). ␤ Note the slight difference in mobility between transfected and endogenous The Phosphorylated P Domain Is Exposed to the Matrix. To deter- PACT, which is caused by the presence/absence of the FLAG tag. (Lower) mine the localization of the phosphorylated, vertebrate-specific Transfected and endogenous PACT lack the N-Term epitope because of N- P␤ domain of PARL, we investigated the topology of the protein. terminal ␤-cleavage. Asterisks indicate nonspecific cleavages. To this end, we used a PARL construct with a hemagglutinin (HA) tag inserted at its N terminus, at position 91, and a FLAG tag at its C terminus (PARL-HA-FCT; Fig. 3C). We perme- Endogenous and Transfected PARL Are Hyperphosphorylated at the abilized PARL-HA-FCT-transfected HeLa cells with increasing Vertebrate-Specific P␤ Domain. To investigate the mechanism of amounts of digitonin, and we performed immunofluorescence regulation of PARL ␤-cleavage in vivo, we conducted mass spec- with antibodies against either the FLAG or HA tag. At low trometric studies to identify posttranslational modifications on the concentrations of digitonin, only the plasma membrane was P␤ domain (14, 15). PARL was immunoprecipitated from lysates permeabilized, allowing the outer membrane receptor Tom20 to

Jeyaraju et al. PNAS ͉ December 5, 2006 ͉ vol. 103 ͉ no. 49 ͉ 18563 Downloaded by guest on October 2, 2021 Fig. 2. Phosphorylation of PARL P␤ domain at Ser-65, Thr-69, and Ser-70 in vivo and in vitro.(A) Phosphorylation of endogenous PARL. LC/MS analysis of PARL from mitochondria purified from human placenta is shown. The protein was immunoprecipitated with the anti-PARL-C-Term antibody (Fig. 1), digested, and subjected to LC/MS analysis. (Left) Molecular ion m/z 1072.932ϩ, which corresponds to a triple-phosphorylated 60VEPRRSDPGTSGEAYKR76 peptide mapping between PARL ␣- and ␤-cleavage sites (Fig. 1A) (11). (Right) Ion m/z 1138.132ϩ, corresponding to the unmodified 77SALIPPVEETVFYPSPYPIR96 peptide, which also maps on the vertebrate-specific P␤ domain of PARL. More than 35% of the mature form of PARL (MAMP; Fig. 1A) could be found through this analysis; the complete list of the ions is shown in Table 1. The identity of each peptide was determined manually and with a Bayesian reconstruction algorithm as well as searching against both theoretical peptide and fragmentation data from the PARL sequence. (B) Phosphorylation of transfected PARL-FCT. LC/MS analysis of transfected PARL-FCT purified from HEK 293 cells is shown. MAMP-FLAG-CTerminus (MAMP-FLAG-CT) was immunoprecipitated with anti-FLAG monoclonal antibody, purified by gel electrophoresis, digested, and analyzed by LC/MS analysis. The triple-phosphorylated 56APRKVEPRRSDPGTSGEAYKR76 peptide, ion m/z 866.393ϩ, is indicated. More than 51% of MAMP sequence could be found through this analysis; the complete list of ions is shown in Table 2. (C) PARL mutant S65A/T69A/S70A is not phosphorylated. LC/MS analysis of transfected PARL-FCT mutant AAA purified from HEK 293 cells is presented. The data show ion m/z 603.32ϩ, corresponding to the unphosphorylated 65ADPGAAGEAYK75 peptide. Note that no phosphorylated peptides encompassing the P␤ domain of this mutant protein were found (data not shown). (D) Tandem MS analysis of phosphorylated PARL. Ion m/z 866.393ϩ was fragmented to detect peptides that, 2ϩ through the loss of phosphate group(s) and/or water (ϪH3PO4), finely map phosphorylation at Ser-65, Thr-69, and Ser-70. The N-terminal ion m/z 686.71 56 ϩ 67 ( APRKVEPRRSDP–H3PO4) and m/z 307.2 ( PGTS–2H3PO4) are shown in (E). The complete list of molecular ions is shown in Table 3. The identity and phosphorylation state of each peptide were determined by both manual interpretation of the spectra and a Mascot search of all of the enhanced product ion scans. (E) Schematic representation summarizing the results showing phosphorylation of endogenous and transfected PARL at residue Ser-65, Thr-69, and Ser-70.

be efficiently recognized by its antibody in Ϸ100% of cells (Fig. not shown), suggesting an additive inhibitory effect of each phos- 3 A and B). As the digitonin concentration increased, first, 70% phorylated amino acid on ␤-cleavage. To demonstrate further the of cells were efficiently labeled with the FLAG along with the inhibitory effect of Asp substitutions on ␤-cleavage, we performed intermembrane space marker cytochrome c. In contrast, the HA a large-scale mutagenesis screening to identify PARL mutants with epitope was efficiently labeled (Ϸ60% of cells) only on treatment constitutive ␤-cleavage. A mutant carrying the 84EETV87 deletion with the highest concentrations of digitonin, which appeared in the P␤ domain, ⌬84–87, showed dramatically increased ␤-cleav- concomitantly with the matrix marker peroxidoreductase age (Fig. 4 B and C). However, in this mutant, Asp substitutions in 3/sp-22 (16). These data indicate that the C-terminal domain of all three phosphorylated residues dominantly reestablished block of PARL is located in the intermembrane space, whereas the ␤-cleavage (Fig. 4D). Notably, none of the PARL mutants analyzed N-terminal phosphorylated P␤ domain is exposed in the mito- in this work with deletions and/or substitutions on the P␤ domain chondrial matrix, thereby correcting our previous examination of had compromised protease activity, as indicated by their ability to PARL topology using immunogold-labeled EM sections (11). cleave in trans a catalytically dead (S277G) PARL protease (Fig. 4E and data not shown). Therefore, lack of ␤-cleavage of the mutant Phosphorylation of the P␤ Domain Regulates ␤-Cleavage. Given the mimicking constitutive phosphorylation of PARL, DDD, is not the proximity of the phosphorylated residues to the ␤-cleavage site, we result of loss of proteolytic activity. This observation also implies investigated whether this modification could have a regulatory that the import and insertion were not compromised, as further function on the cleavage itself. We therefore mutated residues confirmed in digitonin-permeabilization experiments (data not Ser-65, Thr-69, and Ser-70 to alanine, to abolish phosphorylation, shown). We conclude that the stable phosphorylation of residues and to aspartic acid, which is commonly used to mimic phosphor- Ser-65, Thr-69, and Ser-70 inhibits PARL ␤-cleavage. ylation by introducing a negative charge (17, 18). We then tested the effect of these mutations on ␤-cleavage, and we found that Asp ␤-Cleavage Mediates PARL Activity in Mitochondrial Morphology. We substitutions of all three phosphorylated amino acids led to strongly next examined the role of ␤-cleavage and phosphorylation on reduced levels of ␤-cleavage (Fig. 4A). Impaired ␤-cleavage was mitochondrial morphology. Transient expression of wild-type also observed with two double Asp mutants, S65D/T69A/S70D and PARL-FCT in HeLa cells resulted in a dramatic increase in S65A/T69D/S70D (Fig. 4A) but not with single substitutions (data mitochondrial fragmentation (Fig. 5 A and B). Similar results were

18564 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604983103 Jeyaraju et al. Downloaded by guest on October 2, 2021 Fig. 3. Localization of PARL P␤ domain in the matrix. (A) HeLa cells were transfected with a construct expressing PARL-HA-FCT (see scheme in C), fixed, and permeabilized with the indicated concentrations of digitonin. For each condition, cells were coimmunostained with anti-FLAG or anti-HA, and anti- Tom20 (to label the outer membrane), or anti-cytochrome c (to label the intermembrane space), or anti-peroxiredoxin 3 (to label the matrix). (B) Quantitation of the experiments shown in A.(C) Scheme summarizing the topology of PARL. Fig. 4. Substitutions mimicking phosphorylation at Ser-65, Thr-69, and Ser-70 inhibit PARL ␤-cleavage. (A) Constructs expressing the indicated mu- observed with the S65A/T69A/S70A mutant and the constitutively tant PARL protein were transfected in either HEK 293 or HeLa cells. The effect ␤ ⌬ of mutations abolishing (Ala) or mimicking (Asp) phosphorylation (17, 18) is -cleaved PARL protein, 84–87. By contrast, similar levels of ␤ expression of the S65D/T69D/S70D mutant (Fig. 5C and data not monitored by the amount of -cleaved form of PARL detected, PACT (Fig. 1A). Note that Asp but not Ala substitutions block ␤-cleavage. IP, immunoprecipi- shown) did not result in significant mitochondrial fragmentation tation; WB, Western blotting. (B) Scheme of the deletions (⌬) within PARL P␤ (Fig. 5), suggesting that transient expression of the nonphospho- ⌬ ␤ domain that have been tested in this work. (C) The 84–87 mutant is consti- rylated, -cleaved form of PARL leads to fragmented mitochon- tutively cleaved at the ␤-cleavage site. (D) Asp substitutions at positions dria. To investigate this observation further, we transfected mutants ⌬ Ser-65, Thr-69, and Ser-70 in the 84–87 dominantly reestablish the block of CELL BIOLOGY ␤ in which -cleavage was abolished by removing (PARL⌬75–79)or ␤-cleavage. (E) Ala and Asp substitutions at the phosphorylated Ser-65, Thr-69, mutating (PARLL79E) the cleavage site. Expression of either pro- and Ser-70 residues do not affect PARL protease activity. HEK 293 cells were tein did not induce mitochondrial fragmentation (Fig. 5 A and B), cotransfected with a catalytically dead PARL protein (PARL-Myc-CT S277G) further indicating that ␤-cleavage is required to initiate this process. and the indicated FLAG-tagged mutant, whose enzymatic activity is moni- Cleavage of PARL at the ␤-site liberates the P␤ peptide, a 25-aa tored by its ability to cleave the inactive PARL in trans and produce PACT (11). peptide that can target the nucleus when released to the cytosol (11). To investigate whether the liberated P␤ peptide is functionally implicated in the initiation of mitochondrial fragmentation, we 20). On the other hand, the role of its mammalian ortholog PARL transfected mutants in which we deleted parts of its sequence, appears less clear. In higher organisms, dynamic changes in mito- ⌬ ⌬ 56–59 and 58–61 (Fig. 4B), and we analyzed the morphology of chondrial shape have been implicated in programmed cell death the mitochondria. Data showed that neither deletion impaired (4–6), a process that emerged late during metazoan evolution. ␤-cleavage and mitochondrial fragmentation (Fig. 5), indicating Therefore, the machinery of mitochondrial fusion and fission is that the function of the P␤ peptide is independent of the initiation of PARL-induced mitochondrial fragmentation. likely to be regulated by mechanisms additional to those for yeast. Our results implicate phosphorylation and cleavage of the P␤ Discussion domain of PARL in mitochondrial morphology. Because this Considerable mechanistic and functional information on the mi- domain is vertebrate-specific (11), this processing apparently is a tochondrial rhomboid protease PCP1P in yeast is available (7, 8, 19, regulatory mechanism that emerged during vertebrate evolution.

Jeyaraju et al. PNAS ͉ December 5, 2006 ͉ vol. 103 ͉ no. 49 ͉ 18565 Downloaded by guest on October 2, 2021 may arise from a complex set of regulators that could be expressed at different levels in different tissues and cells. To date, the only reversible phosphorylation/dephosphorylation events known to occur within the mitochondrial intermembrane space or matrix compartment are limited to the E1 subunits of the pyruvate and branched-chain ␣-ketoacid dehydrogenase complexes (24, 25). The identification of the kinase/phosphatase couple that regulates PARL cleavage will be of critical importance to under- stand the regulation of mitochondrial morphology in different tissues. Moreover, the discovery of a role for phosphorylation in mitochondrial dynamics could also provide an explanation for the contrasting reports on the effect of proteins controlling mitochon- drial dynamics. For example, expression of OPA1 increased mito- chondrial fusion in mouse embryonic fibroblasts and in NIH 3T3 fibroblasts, whereas it resulted in dramatic fragmentation in COS-7 cells (23, 26, 27). Similarly, expression of DRP1 did not lead to fission in most cell types, but it was reported to fragment organelles in an inducible HeLa cell line (28). Finally, it is tempting to speculate that tissue selectivity of the clinical phenotype of domi- nant optic atrophy and Charcot–Marie–Tooth IIa, caused by mu- tations in OPA1 and MFN2 respectively, could similarly be a consequence of differential phosphorylation (6). ␤-Cleavage liberates a 25-aa nuclear-targeted peptide termed P␤ peptide (11). Topology of PARL now shows that this peptide is generated in the matrix. A recent MS study has demonstrated the existence of a constant efflux of a large number of peptides from the mitochondria (29). These peptides, which originate from the cleavage of proteins localized mainly in the matrix and inner membrane, range in size from 6 to 27 aa, and they are extruded to the cytosol in an ATP- and temperature-dependent manner (29). Whether the P␤ peptide can be exported from the matrix to the cytosol remains to be demonstrated. However, the existence of specialized machinery for the export of matrix peptides of similar size supports this possibility. The fact that an integral P␤ sequence is not required for PARL-mediated mitochondrial fragmentation (Fig. 5A) is consistent with the possibility that the P␤ peptide mediates mitochondria-to-nucleus signaling (11).

Fig. 5. Cleavage of the vertebrate-specific P␤ domain is required to mediate Materials and Methods PARL activity in mitochondrial morphology. (A) HeLa cells were transfected Cell Lines and Antibodies. HEK 293 and HeLa cells were purchased with the indicated PARL-FCT constructs, fixed, permeabilized, and stained from American Type Culture Collection (Manassas, VA) and with monoclonal anti-FLAG (green) and polyclonal anti-Tom20 (red). Images maintained under standard cell culture conditions. Cells were were taken with the Olympus FV1000 confocal microscope. (Scale bars, 5 ␮m.) transfected at 40% confluence with FuGENE 6. The antibodies (B) The mitochondrial morphologies of the wild-type and PARL mutants used in this work were: polyclonal anti-GFP (Clontech, Mountain shown in A were quantified from three independent experiments, counting View, CA), monoclonal anti-GFP (Invitrogen, Carlsbad, CA), 100 cells per experiment. (C) Cells in A express similar levels of transfected proteins. Anti-FLAG immunoprecipitation (IP) and Western blot (WB) analysis monoclonal anti-cytochrome c and polyclonal anti-DsRed (PharM- of the FLAG-tagged PARL constructs used in A are shown. Equal amounts of ingen, San Diego, CA), monoclonal anti-HA (Covance, Denver, immunoprecipitated protein were loaded. Note that in separate experiments, PA), mouse M2 monoclonal and rabbit polyclonal anti-FLAG. we verified that the immunoprecipitation efficiently depleted all of the Polyclonal antibodies against the matrix marker peroxiredoxin 3 transfected protein, validating this comparison (data not shown). (30) were raised in rabbits against the recombinant human GST- PRDX3 protein following standard immunization protocols. Se- rum was tested for specificity by preadsorption with the antigen. Recently, it has been shown that deletion of Parl in the mouse Anti-PARL-N-Term antibody has been already described in ref. 11. resulted in premature postnatal death (21), which correlated with Anti-PARL-C-Term was raised against a peptide spanning the last reduced levels of cleaved OPA1. OPA1 has been shown to be 12 aa of PARL conjugated to keyhole limpet hemocyanin, accord- involved in the regulation of the so-called ‘‘cristae remodeling’’ ing to standard immunization protocols. Immunoprecipitations pathway of apoptosis (21, 22) and the regulation of mitochondrial done with this antiserum were performed by covalently coupling fusion (23). Although PARL was shown to cleave OPA1, only this antiserum to protein A-agarose beads. minor changes in mitochondrial morphology were observed in Ϫ Ϫ Parl / fibroblasts (21), which indicates that PARL is not directly Constructs. The vector used to express the human PARL protein required for mitochondrial fusion (21). Therefore, the expression of in mammalian cells was pcDNA3. The PARL-FCT and PARL- a cleaved form of PARL appears to be a gain of function, leading HA-FCT constructs have been described in ref. 11. Mutants of to the fragmented morphology we have observed in this work. these PARL constructs were obtained by site-directed mutagen- Because PARL-induced fragmentation depends on its phosphor- esis. All mutations were confirmed by DNA sequencing. ylation and cleavage, the functional outcome of PARL expression on steady-state mitochondrial morphology is likely to be regulated PARL Cleavage Analysis. Cells were transfected with the indicated by the abundance and activity of the yet-unidentified PARL kinase PARL construct, grown for 24–36 h (unless otherwise indi- phosphatase, and protease. Thus, apparently disaccording results cated), washed with Dulbecco’s PBS, and lysed in RIPA buffer

18566 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0604983103 Jeyaraju et al. Downloaded by guest on October 2, 2021 containing a mixture of protease inhibitors and 1 mM sodium pass filter. Acquired images and multichannel overlaying were done orthovanadate. Immunoprecipitations were performed with with TillVision IV software (TILL Photonics, Pleasanton, CA). anti-FLAG-M2 monoclonal antibody or anti-PARL-C-Term at Quantification of mitochondrial morphology was done according to 4°C overnight as described in ref. 31. Immunocomplexes were mitochondrial length to width ratio. When the ratio was Ͼ3:1, the washed four times with RIPA and denatured at 85°C for 4 min mitochondria were classified as tubular and rod-shaped, and when in Laemmli buffer. Samples were fractionated by SDS/PAGE on the ratio was Ͻ3:1, mitochondria were classified as fragmented. a 4–12% (wt/vol) 2-[bis(2-hydroxyethyl)amino]-2-(hydroxy- Data were obtained from 100 cells from each condition, and methyl)1,3-propanediol gel, blotted on PVDF membranes, im- standard deviations were calculated from three independent ex- munodetected by Western blot analysis, and imaged by using the periments. High-resolution images were obtained from samples Versadoc system (Bio-Rad, Hercules, CA). For MS analysis, gels transfected with PARL-FCT constructs and costained with mono- were stained with colloidal Coomassie blue. Stained bands were clonal anti-FLAG and polyclonal anti-Tom20 by using an Olympus excised and stored at Ϫ70°C until ready for LC/MS analysis. FV1000 confocal microscope (Olympus Canada). A 100X U Plan Apochromat objective NA 1.45 was used along with the argon laser MS. Samples were prepared as described in ref. 15. For capillary to excite the 488 nm secondary Alexa 488 and the Red HeNe laser LC/MS, samples were analyzed by directed infusion of chromato- for Alexa 647 secondary antibody. The images shown are from graphically separated components on an MDS Sciex QStar XL 10–20 compressed Z stacks taken in 0.12-␮m intervals to capture mass spectrometer (Applied Biosystems, Foster City, CA) inter- Ϸ2-␮m sections of the cell by using Kalman averaging of two scans faced with an Ultimate micropump (LC Packings, Sunnyvale, CA). each (33). Capillary columns (150 ␮m ϫ 100 mm) were packed in-house with Digitonin permeabilization. Digitonin was recrystallized as described Majic C-18 reversed-phase packing material. A 150-min continuous in ref. 34 and resuspended in PBS. Transfected cells were fixed in gradient was used for separation with buffer A [2% ACN (vol/vol)/ 4% paraformaldehyde and permeabilized with increasing concen- 0.1% formic acid/0.01% TFA] followed by buffer B [10% isopropyl trations of digitonin before standard immunofluorescence was alcohol (vol/vol)/80% ACN (vol/vol)/0.1% formic acid/0.01% performed with the indicated antibody as described previously. The TFA]. Dried samples were resuspended in A buffer and loaded data were quantified from 100 cells in three independent experi- directly on the capillary column. Samples were sprayed at 4500V ments (35). and MS along with tandem MS data were acquired on the fly by using the Analyst QS software (Applied Biosystems). Isolation of Mitochondria. Fresh human placenta was obtained with appropriate permission, cut into fragments, and washed with PBS Data Analysis. Resultant data were reconstructed manually and with before decanting into 2 volumes per volume of mitochondrial a Bayesian reconstruction algorithm, and they were searched isolation buffer (220 mM mannitol/68 mM sucrose/20 mM Hepes, against both theoretical peptide and fragmentation data from the pH 7.4/80 mM KCl/0.5 mM EGTA/2 mM MgOAc/protease inhib- PARL sequence. Tandem data were used for web-based searches itors). The tissue was homogenized by using a Waring blender (Cole with Mascot (Matrix Science Ltd., London, U.K.). Matching tan- Palmer, Ansou, QC, Canada), and mitochondria were isolated by dem data were verified manually. standard differential centrifugation. The mitochondrial pellet was resuspended in 100 ml of mitochondrial isolation buffer with 10% Mitochondrial Morphology Analysis. Fluorescence imaging. HeLa cells glycerol, then it was snap frozen and stored at Ϫ80°C. were transfected as described in ref. 32. To stain mitochondria with potentially sensitive dyes, cells were incubated with 50 nM Mit- We thank Dr. Gordon Shore (McGill University, Montreal, QC, Canada) oFluor Red 589 (Invitrogen) at 37°C for 15 min before imaging. For for the anti-Tom20 antibody and Dr. Jordan Fishman (21st Century quantification of mitochondrial phenotypes, images were obtained Biochemicals) for assistance in the generation of antibodies. This work was with an Olympus IX70 microscope (Olympus Canada, Markham, supported by grants from the Natural Sciences and Engineering Research ϫ Council of Canada, the Canada Foundation for Innovation, and the Centre ON, Canada) through a 100 objective U Plan Apochromat, NA de Recherche Universite´Laval Robert-Giffard (to L.P.); grants from the 1.35–0.50 objective, excited at 500 nm (yellow fluorescent protein; Canadian Institutes of Health Research and the Canada Foundation for YFP), 434 nm (cyan fluorescent protein; CFP), and 589 nm Innovation (to the H.M.M. laboratory); a Fonds de la Recherche en Sante´ (MitoFluor Red 589) with the Polychrome IV monochrometer. du Que´becJunior-2 Scholarship (to L.P.); and a Canadian Institutes of The emitted light was filtered through a triple CFP/YFP/DsRed Health Research New Investigator Award (to H.M.M.).

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