Abstract
Dolichol-phosphate mannose (DPM) synthase is required for synthesis of the glycosylphosphatidylinositol (GPI) anchor, N-glycan precursor, protein O-mannose, and C-mannose. We previously identified DPM3, the third component of this enzyme, which was co-purified with DPM1 and DPM2. Here, we have established mutant Chinese hamster ovary (CHO) 2.38 cells that were defective in DPM3. CHO2.38 cells were negative for GPI-anchored proteins, and microsomes from these cells showed no detectable DPM synthase activity, indicating that DPM3 is an essential component of this enzyme. A coiled-coil domain near the C terminus of DPM3 was important for tethering DPM1, the catalytic subunit of the enzyme, to the endoplasmic reticulum membrane and, therefore, was critical for enzyme activity. On the other hand, two transmembrane regions in the N-terminal portion of DPM3 showed no specific functions. DPM1 was rapidly degraded by the proteasome in the absence of DPM3. Free DPM1 was strongly associated with the C terminus of Hsc70-interacting protein (CHIP), a chaperone-dependent E3 ubiquitin ligase, suggesting that DPM1 is ubiquitinated, at least in part, by CHIP.
Highlights
O-mannosylation and C-mannosylation that occur in the endoplasmic reticulum (ER) lumen have been shown to be Dolichol-phosphate mannose (DPM)-dependent [9, 10]
Our previous analyses using mammalian cells revealed that: (i) DPM2 stabilizes the expression of DPM3; (ii) DPM3 binds both DPM2 and DPM1; (iii) the N-terminal transmembrane regions of DPM3 are important for binding to DPM2; and (iv) the C-terminal hydrophilic region of DPM3 is important for binding to DPM1 [15, 16]
DPM3 Is Essential for DPM Synthase Activity—We previously identified DPM3 in the DPM synthase complex that was affinity-purified using DPM1 double-tagged with GST and FLAG [16]
Summary
O-mannosylation and C-mannosylation that occur in the ER lumen have been shown to be DPM-dependent [9, 10]. DPM is synthesized from dolichol phosphate and GDP-Man on the cytosolic surface of the ER membrane by DPM synthase (GDP-␣-Man: dolichol-phosphate -mannosyltransferase; EC 2.4.1.83) and is flipped onto the luminal side and used as a donor substrate. In lower eukaryotes, such as Saccharomyces cerevisiae and Trypanosoma brucei, DPM synthase consists of a single component (Dpm1p and TbDpm, respectively) that possesses one predicted transmembrane region near the C terminus for anchoring to the ER membrane [11, 12]. We describe the essential role of the C-terminal coiled-coil domain of DPM3 and the fate of DPM1 in DPM3-defective mutant cells
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