Mutant COMP is retained in the endoplasmic reticulum of chondrocytes. (a–f) Chondrocytes transduced with 20 MOI (a and b) or 200 MOI (c–f) were cultured in alginate for two weeks before ultrastructural analysis. Low-level expression of mutant COMP (a) leads to a marked distention of the ER (arrows). (b) Magnification of a. (c) A cell with a high-level overexpression of wild-type COMP exhibits a normal ER (arrow) with occasional formation of granular structures (arrowhead). L, lipid droplets. Chondrocytes overexpressing mutant COMP reveal multiple granular structures (d) that are surrounded by a membrane carrying ribosomes, thus corresponding to granular inclusions in the ER (arrow). (e) Magnification of d. Immunogold-labeling shows that the inclusions contain COMP (f). M, mitochondriae. Bars in a, c, d, 1 μm; b and f, 200 nm; e, 100 nm. (g) Cell lysates (Ly) and supernatants (Su) from transfected pulse-labeled monolayer cultures were digested with N-glycosidase F (N-Glc-F), endoglycosidase H (EndoH), or left untreated after c-myc specific precipitation. Molecular weight standards are indicated on the left side of the gels, recombinant COMP on the right side. The arrows mark origin and front of the separating gel. (h) Lysates of transduced monolayer cultures were precipitated with antibodies against Grp78 or calreticulin (Calret) and immunoblotted. Mu, mutant; Wt, wild-type COMP expressing cultures.