Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells.
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 vitroenzymatic 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.


CONCLUSIONS
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.
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.
Jeffrey S
JS
Rush
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.
Thangaiah
T
Subramanian
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.
Karunai Leela
KL
Subramanian
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.
Fredrick O
FO
Onono
Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.
Charles J
CJ
Waechter
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.
H Peter
HP
Spielmann
Click chemistry; dolichol; flippases; photo-affinity probes; proteomics