Self-regulated Cleavage of the Mitochondrial Intramembrane-cleaving Protease PARL Yields Pβ, a Nuclear-targeted Peptide

Regulated intramembrane proteolysis (RIP) is an emerging paradigm in signal transduction. RIP is mediated by intramembrane-cleaving proteases (I-CliPs), which liberate biologically active nuclear or secreted domains from their membrane-tethered precursor proteins. The yeast Pcp1p/Rbd1p protein is a Rhomboid-like I-CliP that regulates mitochondrial membrane remodeling and fusion through cleavage of Mgm1p, a regulator of these essential activities. Although this ancient function is conserved in PARL (Presenilins-associated Rhomboid-like protein), the mammalian ortholog of Pcp1p/Rbd1p, the two proteins show a strong divergence at their N termini. However, the N terminus of PARL is significantly conserved among vertebrates, particularly among mammals, suggesting that this domain evolved a distinct but still unknown function. Here, we show that the cytosolic N-terminal domain of PARL is cleaved at positions 52-53 (α-site) and 77-78 (β-site). Whereas α-cleavage is constitutive and removes the mitochondrial targeting sequence, β-cleavage appears to be developmentally controlled and dependent on PARL I-CliP activity supplied in trans. The β-cleavage of PARL liberates Pβ, a nuclear targeted peptide whose sequence is conserved only in mammals. Thus, in addition to its evolutionarily conserved function in regulating mitochondrial dynamics, PARL might mediate a mammalian-specific, developmentally regulated mitochondria-to-nuclei signaling through regulated proteolysis of its N terminus and release of the Pβ peptide. Regulated intramembrane proteolysis (RIP) is an emerging paradigm in signal transduction. RIP is mediated by intramembrane-cleaving proteases (I-CliPs), which liberate biologically active nuclear or secreted domains from their membrane-tethered precursor proteins. The yeast Pcp1p/Rbd1p protein is a Rhomboid-like I-CliP that regulates mitochondrial membrane remodeling and fusion through cleavage of Mgm1p, a regulator of these essential activities. Although this ancient function is conserved in PARL (Presenilins-associated Rhomboid-like protein), the mammalian ortholog of Pcp1p/Rbd1p, the two proteins show a strong divergence at their N termini. However, the N terminus of PARL is significantly conserved among vertebrates, particularly among mammals, suggesting that this domain evolved a distinct but still unknown function. Here, we show that the cytosolic N-terminal domain of PARL is cleaved at positions 52-53 (α-site) and 77-78 (β-site). Whereas α-cleavage is constitutive and removes the mitochondrial targeting sequence, β-cleavage appears to be developmentally controlled and dependent on PARL I-CliP activity supplied in trans. The β-cleavage of PARL liberates Pβ, a nuclear targeted peptide whose sequence is conserved only in mammals. Thus, in addition to its evolutionarily conserved function in regulating mitochondrial dynamics, PARL might mediate a mammalian-specific, developmentally regulated mitochondria-to-nuclei signaling through regulated proteolysis of its N terminus and release of the Pβ peptide. Regulated intramembrane proteolysis, RIP, 1The abbreviations used are: RIP, regulated intramembrane proteolysis; GFP, green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin A; HEK 293, human embryonic kidney 293 (cells); I-CliP, intramembrane-cleaving protease; PARL, Presenilins-associated Rhomboid-like (protein); MAMP, mature mitochondrial PARL; MES, 2-(N-morpholino)ethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; PARL-F, FLAG-tagged PARL; PARL-HF, FLAG-tagged PARL with HA tag; MTS, mitochondrial targeting sequence; NLS nuclear localization signal; PACT, PARL C-terminal (protein); PNT, PARL N-terminal peptide; TMH, transmembrane helix. APP, amyloid-β precursor protein; AID/AICD, APP intracellular domain; RHO, Rhomboid is a signaling mechanism that involves the generation of biologically active peptides from membrane-tethered precursor proteins (1Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar). RIP was originally described as a mechanism requiring two sequential cleavages carried out by distinct proteases (2Brown M. Goldstein J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar). The first protease cleaves the precursor protein close to a transmembrane helix (TMH), whereas the second cleavage, executed by an intramembrane-cleaving protease, or I-CliP, occurs within the TMH and releases a biologically active peptide. In the well characterized case of the sterol regulatory element-binding protein (SREBP), cleavage by the I-CliP site-2 protease (3Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar) results in the liberation of a nuclear transcription factor (2Brown M. Goldstein J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11041-11048Crossref PubMed Scopus (1110) Google Scholar, 3Rawson R.B. Zelenski N.G. Nijhawan D. Ye J. Sakai J. Hasan M.T. Chang T.Y. Brown M.S. Goldstein J.L. Mol. Cell. 1997; 1: 47-57Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar). A similar process has also been demonstrated for Presenilin-1 (PS1) (4Tandon A. Fraser P. Genome Biol. 2002; 3 (Reviews3014)Crossref PubMed Google Scholar). Intramembrane cleavage of Notch and APP by PS1 results in the liberation of the intracellular domains of these proteins, NICD and AID/AICD, respectively (5De Strooper B. Annaert W. Cupers P. Saftig P. Craessaerts K. Mumm J.S. Schroeter E.H. Schrijvers V. Wolfe M.S. Ray W.J. Goate A. Kopan R. Nature. 1999; 398: 518-522Crossref PubMed Scopus (1808) Google Scholar, 6Passer B. Pellegrini L. Russo C. Siegel R.M. Lenardo M.J. Schettini G. Bachmann M. Tabaton M. D'Adamio L. J. Alzheimers Dis. 2000; 2: 289-301Crossref PubMed Scopus (203) Google Scholar), which are targeted to the nucleus and regulate gene expression (7Haines N. Irvine K.D. Nat. Rev. Mol. Cell Biol. 2003; 4: 786-797Crossref PubMed Scopus (338) Google Scholar, 8Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1058) Google Scholar). The recent characterization of Rhomboid-1, a previously described regulator of Drosophila development (9Bier E. Jan L.Y. Jan Y.N. Genes Dev. 1990; 4: 190-203Crossref PubMed Scopus (345) Google Scholar, 10Ruohola-Baker H. Grell E. Chou T.B. Baker D. Jan L.Y. Jan Y.N. Cell. 1993; 73: 953-965Abstract Full Text PDF PubMed Scopus (127) Google Scholar), has brought new insights into RIP. Indeed, Rhomboid-1 is an I-CliP that does not require a preliminary cleavage of its substrate, Spitz, and the single cleavage, which occurs inside a TMH, yields a secreted regulatory peptide (11Urban S. Lee J.R. Freeman M. Cell. 2001; 107: 173-182Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar). The Rhomboid family of serine I-CliPs consists of two subfamilies, denoted the RHO and PARL subfamilies (12Koonin E.V. Makarova K.S. Rogozin I.B. Davidovic L. Letellier M.C. Pellegrini L. Genome Biol. 2003; 4: R19Crossref PubMed Google Scholar), the latter being named after the Presenilins-associated Rhomboid-like protein (13Pellegrini L. Passer B.J. Canelles M. Lefterov I. Ganjei J.K. Fowlkes B.J. Koonin E.V. D'Adamio L. J. Alzheimers Dis. 2001; 3: 181-190Crossref PubMed Scopus (55) Google Scholar). The conserved core of the Rhomboid family consists of six transmembrane helices. Bacterial and archaeal proteins of this family contain only the six conserved TMHs, whereas most of the eukaryotic members have an additional TMH that is located either upstream (PARL subfamily) or downstream (RHO subfamily) of the core (12Koonin E.V. Makarova K.S. Rogozin I.B. Davidovic L. Letellier M.C. Pellegrini L. Genome Biol. 2003; 4: R19Crossref PubMed Google Scholar). The yeast member of the PARL subfamily, Pcp1p/Rbd1p, is a mitochondrial I-CliP that appears to regulate different mitochondrial activities through the cleavage of the dynamin-like GTPase Mgm1p, an essential mediator of mitochondrial fusion, and Ccp1, a protein required for peroxide and toxic radical scavenging (14Esser K. Tursun B. Ingenhoven M. Michaelis G. Pratje E. J. Mol. Biol. 2002; 323: 835-843Crossref PubMed Scopus (141) Google Scholar, 15Herlan M. Vogel F. Bornhovd C. Neupert W. Reichert A.S. J. Biol. Chem. 2003; 278: 27781-27788Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 16McQuibban G.A. Saurya S. Freeman M. Nature. 2003; 423: 537-541Crossref PubMed Scopus (314) Google Scholar). PARL, the human ortholog of Pcp1p/Rbd1p, can rescue the phenotype of a yeast strain lacking Pcp1p/Rbd1p activity, therefore indicating that the role of the members of the PARL subfamily in mitochondrial protein processing is conserved in eukaryotes (16McQuibban G.A. Saurya S. Freeman M. Nature. 2003; 423: 537-541Crossref PubMed Scopus (314) Google Scholar). However, Pcp1p/Rbd1p and PARL share no significant conservation in their N-terminal regions, whereas in vertebrates, particularly in mammals, this part of the protein is strongly conserved (12Koonin E.V. Makarova K.S. Rogozin I.B. Davidovic L. Letellier M.C. Pellegrini L. Genome Biol. 2003; 4: R19Crossref PubMed Google Scholar). This suggests that the N-terminal portion of mammalian PARL could have a distinct, unknown function. Here, we show that the N-terminal domain of PARL is cleaved in two unique sites. The first cleavage is constitutive and removes the mitochondrial import peptide, whereas the second cleavage, which depends on the I-CliP activity of PARL supplied in trans and is developmentally regulated, liberates Pβ, a nucleus-targeted peptide. Thus, the PARL case represents a new type of RIP in which the putative signaling moiety is part of the I-CliP itself. Cell Lines and Transfections—Cell lines HEK 293, neuro-2a (N2a), HeLa, and COS-1 were obtained from the American Type Culture Collection and grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mm glutamine, 10 μg/ml streptomycin, and 10 μg/ml penicillin. Cells were transfected at 20-40% of confluence with FuGENE6 (Roche). Cortical primary cultures were prepared from newborn brain of rats as described (17De Koninck P. Cooper E. J. Neurosci. 1995; 15: 7966-7978Crossref PubMed Google Scholar). Immunoblot and Immunoprecipitation Assays—Forty-eight to seventy-two hours after transfection, cells were lysed in lysis buffer (50 mm Tris, 150 mm NaCl, 2 mm EDTA, and 1% Nonidet P-40, pH 7.6) containing a proteinase inhibitor mixture (Roche Applied Science). Lysate was spun at 10,000 × g for 10 min, and the supernatant was recovered for immunoblot or immunoprecipitation analysis. For immunoprecipitation analysis, an antibody was added, and the immunoprecipitation reaction was incubated at 4 °C for 12 h with gentle rocking. Immunocomplexes were captured by the addition of protein A/G-agarose beads (Pierce) and incubation for 2 h at room temperature. Beads were washed three times in wash buffer I (50 mm Tris, 500 mm NaCl, 2 mm EDTA, and 0.2% Nonidet P-40, pH 7.6), washed three times in wash buffer II (50 mm Tris, 150 mm NaCl, 2 mm EDTA, 0.2% Nonidet P-40, and 0.1% SDS, pH 7.6), and resuspended in 100 μl of Laemmli loading buffer. Samples were heated at 85 °C for 2 min and separated on a 4-12% polyacrylamide-SDS gel (Invitrogen). Proteins were blotted onto nitrocellulose membranes and probed with the specified antibodies as described (18Pellegrini L. Passer B.J. Tabaton M. Ganjei J.K. D'Adamio L. J. Biol. Chem. 1999; 274: 21011-21016Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 19Rossi V. Motto M. Pellegrini L. J. Biol. Chem. 1997; 272: 13758-13765Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Immunoblots were developed using the SuperSignal system (Pierce). Mitochondria Protease Protection Assay—Mitochondria-enriched preparations were obtained from one Petri dish containing confluent HEK 293 cells transfected with the PARL-HF construct (FLAG-tagged PARL with an HA tag). Cells were washed in ice-cold Dulbecco's modified phosphate-buffered saline and disrupted with 10-20 strokes of a Teflon-coated Dounce homogenizer in 0.6 ml of homogenization buffer, which consisted of 0.1 m sucrose, 10 mm MES, 10 mm Hepes, pH 7, 100 mm KCl, 10 mm KH2PO4, 3.5 mm MgCl2, and 1 mm EGTA. Mitochondria-enriched fractions were obtained by differential centrifugation. The pellet was resuspended in 100 μl of homogenization buffer and 20-μl aliquots subjected to proteinase-K or trypsin (Sigma) digestion (1 μg) for 30 min at room temperature. Antibody Preparation—For the preparation of the anti-PNT antibody (directed against a PARL peptide located near the N terminus), a 12-amino acid-long peptide spanning amino acids 54-66 of PARL was synthesized, purified by to bovine and used to to for 2 and was used and and for and The antibody used for was obtained by peptide of the and used in The of the anti-PNT was by the with a N-terminal fusion was the antibody was incubated h at 4 °C with of cells a N-terminal of fusion expression we used the whereas for mammalian expression we used the and the or that the used in this was at the downstream of the This was to of with fusion proteins a sequence this The amino sequence by the FLAG-tagged PARL construct in the of the PARL terminus is peptide is The amino sequence by the PARL-HF construct in the of PARL is peptide is N-terminal of proteins by HEK 293 cells were transfected with the construct cells were lysed in buffer (50 mm pH 1% 1% 0.1% SDS, 1 mm EDTA, and 150 mm in the of a protease inhibitor mixture (Roche Applied Science). Cell was with beads and resuspended in Laemmli buffer. proteins were on a 4-12% blotted on and by with a to of the and proteins were from the and their N-terminal were on an Applied protein 1 of the protein was on the A using was for The were by with and on a system at the of a sequence analysis. primary cultures were in and 0.1 m buffer and incubated in and or nuclear protein and using antibodies HEK 293 cells were and washed in for the in For analysis, cells were incubated with and and as described For analysis, cells were incubated with by antibody incubation and as described A. N. A. I. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). were using and using a 12 PARL N at the through a the role of the PARL N-terminal a of HEK 293 cells transfected with a construct was with antibodies against a PARL peptide located near the N terminus and with antibodies against the the anti-PNT two of and were we that these to the PARL and to the with the mitochondrial targeting sequence named this second of the PARL protein MAMP, after mature mitochondrial PARL, and the cleavage that was PARL in the to be an as by the of PARL a protein of was with the The of this protein was by of the amino that the of Rhomboid-like proteases A and (11Urban S. Lee J.R. Freeman M. Cell. 2001; 107: 173-182Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, E.V. Makarova K.S. Rogozin I.B. Davidovic L. Letellier M.C. Pellegrini L. Genome Biol. 2003; 4: R19Crossref PubMed Google Scholar, 16McQuibban G.A. Saurya S. Freeman M. Nature. 2003; 423: 537-541Crossref PubMed Scopus (314) Google Scholar). In the cleavage of the putative MTS, which MAMP, was not by the the of the and in the used for these we that the was the of a cleavage of the PARL N terminus this PARL PACT, after PARL and the cleavage that was denoted of at h in HEK 293 cells results were obtained in the but not in the COS-1 or in was in these lines whereas α-cleavage occurs β-cleavage depends on the I-CliP activity of PARL and might be regulated, in a the and β-cleavage cells were transfected with the construct containing an HA upstream of PARL-HF was in that were with the and but not with the anti-PNT this the β-cleavage the and HA the cleavage of PARL and were and subjected to N-terminal by The was located amino acids and and the was amino acids and The is within a sequence that is conserved in of PARL, whereas the is conserved only in a of the is in in and in Drosophila Thus, whereas α-cleavage is with mitochondrial import of PARL and is conserved in β-cleavage is to be a of the sequence of we the in positions to as a an upstream of amino acids the in the β-cleavage the generation of PACT, whereas the no into the mechanism of the cleavage of PARL, we HEK 293 cells with the PARL-HF and the or the The of from PARL-HF was in the of PARL but not in that of the and not indicating that the activity of PARL required for the β-cleavage of its N terminus could be supplied in trans. that β-cleavage is either executed by an unknown protease that is a cleavage or by PARL through an However, the latter appears to be a the is not within a TMH, an essential in RIP (1Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar, A. Fraser P. Genome Biol. 2002; 3 (Reviews3014)Crossref PubMed Google Scholar, S. Lee J.R. Freeman M. Cell. 2001; 107: 173-182Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar, 15Herlan M. Vogel F. Bornhovd C. Neupert W. Reichert A.S. J. Biol. Chem. 2003; 278: 27781-27788Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 16McQuibban G.A. Saurya S. Freeman M. Nature. 2003; 423: 537-541Crossref PubMed Scopus (314) Google Scholar). of PARL Pβ, a RIP signaling cytosolic of the cleaved precursor proteins B. Pellegrini L. Russo C. Siegel R.M. Lenardo M.J. Schettini G. Bachmann M. Tabaton M. D'Adamio L. J. Alzheimers Dis. 2000; 2: 289-301Crossref PubMed Scopus (203) Google Scholar) are into the nucleus regulate gene expression (1Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar, N. Irvine K.D. Nat. Rev. Mol. Cell Biol. 2003; 4: 786-797Crossref PubMed Scopus (338) Google Scholar, 8Cao X. Sudhof T.C. Science. 2001; 293: 115-120Crossref PubMed Scopus (1058) Google Scholar). we that the N-terminal peptide from the β-cleavage of PARL could be a nucleus-targeted this we transfected HEK 293 and cells with different of the N-terminal domain of PARL or to of HEK 293 cells transfected with a construct mitochondrial localization of this suggesting that the mitochondrial targeting sequence of PARL is located within the spanning the first amino acids and the conserved α-cleavage of PARL N terminus This is to have an which is of mitochondrial targeting and is as an using the 1995; Google Scholar). targeting was in that at the N indicating that the N-terminal of the is In a construct that the the and β-cleavage which is conserved only in vertebrates, a nuclear localization and and nuclear localization are at the N and of nuclear proteins and their function is by their within a fusion protein also in A. Rev. 2001; PubMed Scopus Google Scholar), we that the the and might an Although this sequence does not contain a or nuclear localization three of amino acids amino acids the first and second of which are conserved in vertebrates, whereas the one is of are of of NLS A. Rev. 2001; PubMed Scopus Google Scholar), which that this sequence of PARL could function as a nuclear localization of the two conserved of amino acids was to a of the nuclear localization of the N terminus and The of these not cells with a nuclear F. D. M.C. J. M. R. Genes Dev. 2003; PubMed Scopus Google Scholar) indicating that these the of nuclear import the of the fusion proteins. that only a peptide by sequential and β-cleavage of PARL has the to be into the that α-cleavage is constitutive and with the of the mitochondrial targeting sequence, whereas the β-cleavage is and to the generation of a nuclear targeting peptide. this nucleus-targeted peptide spanning amino acids of PARL Pβ is to be a acid-long nuclear peptide by β-cleavage of PARL β-cleavage of PARL, we on primary cultures using an antibody against a peptide a sequence located the and β-cleavage and This antibody by analysis, PARL-HF in the of transfected HEK 293 cells in the of this In in the of transfected HEK 293 cells anti-PNT was in the and the A and nuclear whereas mitochondrial The of was by of well as the of the and the P. 2002; PubMed Scopus Google Scholar) as well as by with and nuclear protein of and respectively PARL is cleaved to Pβ, which is targeted to the nucleus or as by the anti-PNT mitochondrial by the of nuclear in Thus, in β-cleavage of PARL appears to be and nuclear of Pβ might be to PARL on the of the nuclear localization of the Pβ peptide to the and localization of PARL in the to Pβ, its could the this HEK 293 cells were transfected with the PARL-HF by analysis. was on the cytosolic of the membrane of the indicating that the N terminus of PARL into the However, the cells were subjected to which mitochondrial and was also in the membrane This with the recent that C. G. N. C. P. L. J. C. E. C. L. B. B. J. Nat. 2000; PubMed Scopus Google Scholar), the mammalian ortholog of and therefore a of PARL, is also in mitochondrial membranes M. N. H. 2003; PubMed Scopus Google Scholar). were by the results of a protease on mitochondrial preparations obtained from HEK 293 This that is to protease digestion β-cleavage on the the of PARL we that β-cleavage occurs at the cytosolic of the mitochondrial membrane and that Pβ is to the from can to the nucleus for the PARL A signal the I-CliP activity of PARL, in the cleavage of either a protease in or a regulator This cleavage in cleaves the N terminus of PARL into the at the the Pβ peptide. Pβ is targeted to the to its nuclear localization in nuclear In this we that the N-terminal domain of PARL is cleaved at two the and at positions 52-53 and Whereas the α-cleavage is constitutive and appears to the of the mitochondrial targeting sequence, which was not previously to is regulated and depends on PARL I-CliP activity supplied in trans. that β-cleavage is not an cleavage of the mitochondrial import peptide but a distinct that appears to with the The β-cleavage Pβ, a peptide that a nuclear localization signal and in is targeted to the This is by the that β-cleavage occurs on the cytosolic of the mitochondrial membrane The of Pβ out the that this peptide has an therefore that Pβ through a with a nuclear peptides can have activity, as 2002; PubMed Scopus Google Scholar) and For is a acid-long peptide that the protein; to from with therefore release and B. D. E. K. S. Nature. 2003; 423: PubMed Scopus Google Scholar). that Pβ in the of this peptide could to of nuclear by with a transcription The of Pβ of a putative new signaling function of PARL, appears to be a case of a gene is in a of for a new function that is to the one J. N. Acad. Sci. PubMed Scopus Google Scholar). the role of in evolved in similar E. G. Nature. 2002; PubMed Scopus Google Scholar), and this to be originally 2003; PubMed Scopus Google Scholar). that the signaling function of PARL is a mammalian as by the conservation of the Pβ NLS as well as its sequence among to a vertebrates, but not and and in the of this The results in addition to its evolutionarily conserved activity in regulating mitochondrial dynamics, mammalian PARL was to a signaling function. in to RIP (1Brown M.S. Ye J. Rawson R.B. Goldstein J.L. Cell. 2000; 100: 391-398Abstract Full Text Full Text PDF PubMed Scopus (1151) Google Scholar, A. Fraser P. Genome Biol. 2002; 3 (Reviews3014)Crossref PubMed Google Scholar), this activity appears to be to Indeed, the release of the Pβ peptide, the putative of the PARL is the activity of a second protease and appears to be has been that I-CliPs can be for proteolysis A. D. Scopus Google Scholar). we show an additional and type of RIP, which involves a cleavage of an I-CliP that results in the release of a peptide. this is the first case of RIP the putative signaling moiety is part of the I-CliP itself. F. for protein P. Koninck for primary Letellier for G. for the PARL in for of the and K. and C. for with

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