Fig. 2.

Phosphorylation of PARL Pβ 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 1072.93²⁺, which corresponds to a triple-phosphorylated ⁶⁰VEPRRSDPGTSGEAYKR⁷⁶ peptide mapping between PARL α- and β-cleavage sites (Fig. 1A) (11). (Right) Ion m/z 1138.13²⁺, corresponding to the unmodified ⁷⁷SALIPPVEETVFYPSPYPIR⁹⁶ peptide, which also maps on the vertebrate-specific Pβ 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-C_Terminus (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 866.39³⁺, 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 603.3²⁺, corresponding to the unphosphorylated ⁶⁵ADPGAAGEAYK⁷⁵ peptide. Note that no phosphorylated peptides encompassing the Pβ domain of this mutant protein were found (data not shown). (D) Tandem MS analysis of phosphorylated PARL. Ion m/z 866.39³⁺ was fragmented to detect peptides that, through the loss of phosphate group(s) and/or water (−H₃PO₄), finely map phosphorylation at Ser-65, Thr-69, and Ser-70. The N-terminal ion m/z 686.71²⁺ (⁵⁶APRKVEPRRSDP–H₃PO₄) and m/z 307.2⁺ (⁶⁷PGTS–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.