FIGURE 5:

ER stress generates LLO-derived lumenal free glycans. (A) Free glycans in PERK^−/− MEFs after treatments with DTT, TG, or TN. The positions of M₅Gn₂ and G₃M₉Gn₂ standards on FACE gels are shown. (B) PERK^−/− MEFs were left untreated, or treated 1 h with either 2 mM DTT or 150 nM TG. ANDS-conjugated free glycans were incubated in the absence or presence of endoglycosidase H or α-mannosidase. Note that Gn₁-ANDS generated by endoglycosidase H runs off the gel. (C) Free glycans released from LLOs in response to M6P are predicted to be in the ER lumen. In contrast, free glycans from protein misfolding, export from the lumen, and enzymatic deglycosylation during ERAD should be cytosolic. In both pathways, glycans can be digested by various exoglycosidases (indicated by the repeating arrows) to generate heterogeneous structures. The lumenal and cytosolic glycan pools can be distinguished by SLO permeabilization. See Figure 1 for explanation of symbols. (D) After treatment of PERK^−/− MEFs with DTT or TG, free glycans were analyzed directly from whole cells (Total), or after permeabilization with SLO, to yield lumenal free glycans (Lum.) in cell bodies and cytosolic free glycans (Cyto.) in permeabilization medium. In this and similar experiments, we noticed that the Total signals appear greater than the sums of the lumenal plus cytosolic signals, presumably due to sample loss occurring during the additional SLO step. (E) PERK^+/+ MEFs, as for (A).