Jeffrey S Rush1, Thangaiah Subramanian1, Karunai Leela Subramanian1, Fredrick O Onono1, Charles J Waechter1, H Peter Spielmann2. 1. Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA. 2. Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA; University of Kentucky College of Medicine, Markey Cancer Center, University of Kentucky, Lexington, Kentucky 40536, USA; Kentucky Center for Structural Biology, University of Kentucky, Lexington, Kentucky 40536, USA; Department of Chemistry, University of Kentucky, Lexington, Kentucky 40536, USA.
Abstract
BACKGROUND: Dolichyl phosphate-linked mono- and oligosaccharides (DLO) are essential intermediates in protein N-glycosylation, C- and O-mannosylation and GPI anchor biosynthesis. While many membrane proteins in the endoplasmic reticulum (ER) involved in the assembly of DLOs are known, essential proteins believed to be required for the transbilayer movement (flip-flopping) and proteins potentially involved in the regulation of DLO synthesis remain to be identified. METHODS: The synthesis of a series of Dol-P derivatives composed of citronellyl-based photoprobes with benzophenone groups equipped with alkyne moieties for Huisgen "click" chemistry is now described to utilize as tools for identifying ER proteins involved in regulating the biosynthesis and transbilayer movement of lipid intermediates. In vitro enzymatic assays were used to establish that the photoprobes contain the critical structural features recognized by pertinent enzymes in the dolichol pathway. ER proteins that photoreacted with the novel probes were identified by MS. RESULTS: The potential of the newly designed photoprobes, m-PAL-Cit-P and p-PAL-Cit-P, for identifying previously unidentified Dol-P-interacting proteins is supported by the observation that they are enzymatically mannosylated by Man-P-Dol synthase (MPDS) from Chinese Hamster Ovary (CHO) cells at an enzymatic rate similar to that for Dol-P. MS analyses reveal that DPM1, ALG14 and several other yeast ER proteins involved in DLO biosynthesis and lipid-mediated protein O-mannosylation photoreacted with the novel probes. CONCLUSION: The newly-designed photoprobes described in this paper provide promising new tools for the identification of yet to be identified Dol-P interacting ER proteins in yeast and mammalian cells, including the Dol-P flippase required for the "re-cycling" of the glycosyl carrier lipid from the lumenal monolayer of the ER to the cytoplasmic leaflet for new rounds of DLO synthesis.
BACKGROUND:Dolichyl phosphate-linked mono- and oligosaccharides (DLO) are essential intermediates in protein N-glycosylation, C- and O-mannosylation and GPI anchor biosynthesis. While many membrane proteins in the endoplasmic reticulum (ER) involved in the assembly of DLOs are known, essential proteins believed to be required for the transbilayer movement (flip-flopping) and proteins potentially involved in the regulation of DLO synthesis remain to be identified. METHODS: The synthesis of a series of Dol-P derivatives composed of citronellyl-based photoprobes with benzophenone groups equipped with alkyne moieties for Huisgen "click" chemistry is now described to utilize as tools for identifying ER proteins involved in regulating the biosynthesis and transbilayer movement of lipid intermediates. In vitro enzymatic assays were used to establish that the photoprobes contain the critical structural features recognized by pertinent enzymes in the dolichol pathway. ER proteins that photoreacted with the novel probes were identified by MS. RESULTS: The potential of the newly designed photoprobes, m-PAL-Cit-P and p-PAL-Cit-P, for identifying previously unidentified Dol-P-interacting proteins is supported by the observation that they are enzymatically mannosylated by Man-P-Dol synthase (MPDS) from Chinese Hamster Ovary (CHO) cells at an enzymatic rate similar to that for Dol-P. MS analyses reveal that DPM1, ALG14 and several other yeast ER proteins involved in DLO biosynthesis and lipid-mediated protein O-mannosylation photoreacted with the novel probes. CONCLUSION: The newly-designed photoprobes described in this paper provide promising new tools for the identification of yet to be identified Dol-P interacting ER proteins in yeast and mammalian cells, including the Dol-P flippase required for the "re-cycling" of the glycosyl carrier lipid from the lumenal monolayer of the ER to the cytoplasmic leaflet for new rounds of DLO synthesis.
Authors: Jonne Helenius; Davis T W Ng; Cristina L Marolda; Peter Walter; Miguel A Valvano; Markus Aebi Journal: Nature Date: 2002-01-24 Impact factor: 49.962
Authors: F Fernandez; J S Rush; D A Toke; G S Han; J E Quinn; G M Carman; J Y Choi; D R Voelker; M Aebi; C J Waechter Journal: J Biol Chem Date: 2001-08-14 Impact factor: 5.157
Authors: Martin Loibl; Lina Wunderle; Johannes Hutzler; Benjamin L Schulz; Markus Aebi; Sabine Strahl Journal: J Biol Chem Date: 2014-02-11 Impact factor: 5.157