Analyses of the Membrane Topology and Physical Interactions of Yeast Dolichyl Phosphate-Glucose Synthase Alg5p

In the biosynthetic pathway of dolichol-linked oligosaccharide (DLO) in the eukaryotic cell, yeast dolichyl-phosphate-glucose synthase (DPGS), Alg5p, catalyzes glucosylation of dolichyl-phosphate (Dol-P) using UDP-glucose, yielding dolichyl-phosphate-glucose (DPG), which is in turn utilized by three luminal glucosyltransferases (Alg6p, Alg8p and Alg10p) as a substrate.In this study, we cloned yeast ALG5 gene encoding DPGS into the vectors for the yeast split-ubiquitin system (YSUS) and explored its membrane topology and physical interaction in detail.Our results indicated that Alg5p possesses two transmembrane domains (TMDs), with both termini orientated toward the cytoplasmic side of the rough ER (rER) membrane and the protein physically interacts with Dpm1p, Sec59p, Cwh8p and Alg7p, which are involved in DLO assembly.In addition, we revealed that the C-terminal loop region (LR4) of Alg5p is essential for the interaction with the Alg7p.