LMAN1 monomer is defective in ER exit. (A) Immunofluorescence staining of the wild-type LMAN1 (WT), ΔHelix, and ΔHM mutants. LMAN1 proteins were detected with a monoclonal anti-Flag antibody. TRAP-α was used as an ER marker. (B) ER exit of wild-type LMAN1, ΔHelix, ΔHM, KKAA, and C466A/C475A mutants. HeLa cells were transfected with the indicated LMAN1 constructs. Membrane from transfected cells were incubated with rat liver cytosol. COPII vesicles were isolated and analyzed for the presence of a resident ER protein, ribophorin I, the endogenous LMAN1, the transfected LMAN1 mutants, and a control cargo protein, SEC22B.