Figure 2.

Oligomeric state of newly synthesized RBP. (A) RBP mRNA was translated in a rabbit reticulocyte lysate system supplemented with dog pancreas microsomes under reducing conditions for 60 min at 27°C. Disulfide oxidation was initiated by the addition of 4 mM GSSG and chased for a further period of 30 and 90 min. At the end of the chase, the microsomes were lysed and the lysate subjected to sedimentation analysis on a 5–25% sucrose gradient. Fractions were collected from the top and precipitated with 10% TCA. The precipitates were analyzed on a 13% nonreducing SDS-PAGE followed by autoradiography. The positions of reduced (Red), intermediate (It), and fully oxidized (Ox) forms are indicated. The pellet fractions are shown at a lighter exposure. (B) To estimate the size of the complex, RBP was subjected to posttranslational folding as described above, and samples taken at 0 and 90 min were sedimented on a tabletop ultracentrifuge using 10–80% glycerol gradient at 50,000 rpm for 4 h. Fractions were collected from the top, TCA precipitated, and analyzed on a 10 or 13% nonreducing SDS-PAGE. Gels (13%) were followed by autoradiography. The positions of the reduced (Red) and fully oxidized (Ox) forms are indicated. Gels (10%) were analyzed by Western blotting using antibodies against ER-chaperones (BiP, PDI, and CNX). (C) Cotranslational oxidation of RBP was performed by translating in presence of GSSG. After 30 and 60 min of initiation of translation, the samples were taken and analyzed on 10–80% glycerol gradient as described above.