FIGURE 1:

UGGT1 increases the solubility of α1-antitrypsin mutants NHK and ATZ. (A–C) The 24-h metabolic labeling of Uggt1^−/− MEFs transfected with (A) NHK, (B) ATZ, or (C) wt-AAT ± UGGT1 cotransfection. Uggt1^−/− MEFs were transfected with expression vectors encoding the misfolded α1-antitrypsin variants NHK or ATZ or wild-type AAT (wt-AAT) and cotransfected with either empty vector or UGGT1 expression vector. Cells were radiolabeled from 24 to 48 h posttransfection in complete medium plus 10 μCi/ml [³⁵S]methionine/cysteine. Extracellular, soluble, and insoluble fractions were produced (Materials and Methods), and TCA-precipitation normalized volumes of each fraction were subjected to double–anti-AAT quantitative IP and PNGaseF digestion and analyzed by reducing SDS–PAGE and autoradiography. Substrate solubility was calculated by dividing total soluble (extracellular + intracellular soluble) substrate amount by insoluble substrate amount. Middle, Student's t test used for statistical testing; right, paired Student's t test used for statistical testing (*p ≤ 0.05). Error bars represent SEM. For ATZ, all UGGT1+ bands were darkened with imaging software to make synthesis (data not shown) ± UGGT1 appear equal.