title: Role of the N-terminal hydrophobic residues of DGKε in targeting the endoplasmic reticulum.
abstract: The endoplasmic reticulum (ER), comprised of an interconnected membrane network, is a site of phospholipid and protein synthesis. The diacylglycerol kinase (DGK) enzyme family catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Both of these lipids are known not only to serve as second messengers but also to represent intermediate precursors of lipids of various kinds. The DGK family is targeted to distinct subcellular sites in cDNA-transfected and native cells. Of DGKs, DGKε localizes primarily to the ER, suggesting that this isozyme plays a role in this organelle. Using experiments with various deletion and substitution mutants, this study examined the molecular mechanism of how DGKε is targeted to the ER. Results demonstrate that the N-terminal hydrophobic sequence 20-40 plays a necessary role in targeting of DGKε to the ER. This hydrophobic amino acid segment is predicted to adopt an α-helix structure, in which Leu22, L25, and L29 are present in mutual proximity, forming a hydrophobic patch. When these hydrophobic Leu residues were replaced with hydrophilic amino acid Gln, the mutant fragment designated DGKε (20-40/L22Q,L25Q,L29Q) exhibits diffuse distribution in the cytoplasm. Moreover, full-length DGKε containing these substitutions, DGKε (L22Q,L25Q,L29Q), is shown to distribute diffusely in the cytoplasm. These results indicate that the N-terminal hydrophobic residues play a key role in DGKε targeting to the ER membrane. Functionally, knockdown or deletion of DGKε affects the unfolding protein response pathways, thereby rendering the cells susceptible to apoptosis, to some degree, under ER stress conditions.
authors.affiliation: Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585, Japan; Department of Otolaryngology, Head and Neck Surgery, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
authors.forename: Hirooki
authors.initials: H
authors.lastname: Matsui
authors.affiliation: Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
authors.forename: Yasukazu
authors.initials: Y
authors.lastname: Hozumi
authors.affiliation: Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Tanaka
authors.affiliation: Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Okada
authors.affiliation: Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Nakano
authors.affiliation: Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585, Japan; Department of Otolaryngology, Head and Neck Surgery, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Suzuki
authors.affiliation: Department of Emergency and Critical Care Medicine, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Iseki
authors.affiliation: Department of Otolaryngology, Head and Neck Surgery, Yamagata University School of Medicine, Yamagata 990-9585, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Kakehata
authors.affiliation: Huntsman Cancer Institute, Department of Oncological Sciences, University of Utah, Salt Lake City, UT 84112, USA.
authors.forename: Matthew K
authors.initials: MK
authors.lastname: Topham
authors.affiliation: Department of Anatomy and Cell Biology, Yamagata University School of Medicine, Yamagata 990-9585, Japan. Electronic address: kgoto@med.id.yamagata-u.ac.jp.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Goto
keywords: Apoptosis; Diacylglycerol kinase; ER stress; Endoplasmic reticulum; Hydrophobicity