FIGURE 5.

Effect of missense mutations on cellular localization of DBH protein. A, PC12 cells expressing wild-type (top) or A348E (bottom) DBH were differentiated with NGF for 4 days and immunostained with anti-Myc antibodies, followed by reaction with an Alexa Fluor 594 chicken anti-rabbit IgG. The same cells were also stained with anti-chromogranin A (CgA) monoclonal antibodies (left) and anti-BiP/GRP78 (KDEL) monoclonal antibodies (right). Wild-type DBH co-localizes with chromogranin A, whereas mutant DBH co-localizes with BiP/GRP78. Scale bars, 10 μm. B, electron microscopic analysis of thin cryosections of PC12 cells expressing wild-type (left) and A348E (right) DBH. A 10-nm immunogold label of wild type is abundantly found on the SG (white arrowhead) and rarely on the ER (black arrowhead). In contrast, immunogold label for mutant DBH is absent from the SG and is mostly found in the ER. Scale bars, 200 nm. C, subcellular fractionation of PC12 cells expressing wild-type and A348E DBH was analyzed by Western blot assay using anti-Myc antibodies (for DBH protein), anti-KDEL (for ER marker), and anti-secretogranin II (for LDCV marker). Wild-type DBH protein was present in fractions 19–23 together with secretogranin, whereas mutant DBH A348E was present in fractions 11–15 together with KDEL.