Fig. 5.

Glycosylation pattern of mutant proteins. A: peptide N-glycosidase F (PNGaseF) or endoglycosidase H (Endo H) deglycosylation of NH₂- and COOH-terminal fragment-tagged mCLCA4 proteins indicated that the 125-kDa full-length protein is immaturely glycosylated (sensitive to both enzymes), whereas the 90- and 40-kDa fragments were only partially sensitive to Endo H, indicating more mature glycosylation. B: the dileucine and diarginine (RxR) mutants demonstrate marked ER or early Golgi trapping since there is no observable 90-kDa fragment, and the 125-kDa protein displays an immature glycosylation pattern. C: mutation of the diarginine motif does not result in ER trapping, since the 90-kDa fragment is detected in the media by immunoprecipitation, indicating highly efficient forward trafficking. Conversely, mutation of the dileucine signals results in complete loss of forward trafficking. D: the AA R⁵⁹²ARAPA⁵⁹⁷ mutant displays a glycosylation pattern consistent with delayed processing, but not ER trapping, since the 90-kDa proteolytic fragment from this mutant, as well as the DD mutant, is resistant to Endo H. Media lanes were by immunoprecipitation.