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Phosphorylation and Cleavage of Presenilin-Associated Rhomboid-Like Protein (PARL) Promotes Changes in Mitochondrial Morphology

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Phosphorylation and Cleavage of Presenilin-Associated Rhomboid-Like Protein (PARL) Promotes Changes in Mitochondrial Morphology 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.
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