title: Septal localization by membrane targeting sequences and a conserved sequence essential for activity at the COOH-terminus of Bacillus subtilis cardiolipin synthase.
abstract: The acidic phospholipid cardiolipin (CL) is localized on polar and septal membranes and plays an important physiological role in Bacillus subtilis cells. ClsA, the enzyme responsible for CL synthesis, is also localized on septal membranes. We found that GFP fusion proteins of the enzyme with NH2-terminal and internal deletions retained septal localization. However, derivatives with deletions starting from the COOH-terminus (Leu482) ceased to localize to the septum once the deletion passed the Ile residue at 448, indicating that the sequence responsible for septal localization is confined within a short distance from the COOH-terminus. Two sequences, Ile436-Leu450 and Leu466-Leu478, are predicted to individually form an amphipathic α-helix. This configuration is known as a membrane targeting sequence (MTS) and we therefore refer to them as MTS2 and MTS1, respectively. Either one has the ability to affect septal localization, and each of these sequences by itself localizes to the septum. Membrane association of the constructs of this enzyme containing the MTSs was verified by subcellular fractionation of the cells. CL synthesis, in contrast, was abolished after deleting just the last residue, Leu482, in the COOH-terminal four amino acid residue sequence, Ser-Pro-Ile-Leu, which is highly conserved among bacterial CL synthases.
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan.
authors.forename: Jin
authors.initials: J
authors.lastname: Kusaka
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Shuto
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan.
authors.forename: Yukiko
authors.initials: Y
authors.lastname: Imai
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Ishikawa
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan.
authors.forename: Tomo
authors.initials: T
authors.lastname: Saito
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan.
authors.forename: Kohei
authors.initials: K
authors.lastname: Natori
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Matsuoka
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hara
authors.affiliation: Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan. Electronic address: koumatsu@mail.saitama-u.ac.jp.
authors.forename: Kouji
authors.initials: K
authors.lastname: Matsumoto
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology*; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D017384:Sequence Deletion; D017855:Transferases (Other Substituted Phosphate Groups) / Q000032:analysis* / Q000235:genetics
chemical_list: D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017855:Transferases (Other Substituted Phosphate Groups); C100091:cardiolipin synthetase
keywords: Bacillus subtilis; CL synthase; ClsA; Membrane targeting sequence; Septal localization