Figure 2.

Asp (D) or Ala (A), but not Pro (P), substitution at PI Tyr-B16 does not affect folding and secretion of PI. A and B: 293 cells transfected with empty vector (EV) or plasmids encoding PI-WT, C(A7)Y, Y(B16)A, Y(B16)D, or Y(B16)P were metabolic labeled for 30 min without chase. The newly synthesized PI was IP by anti-insulin followed by analysis of tris-tricine-urea-SDS-PAGE under both nonreducing and reducing conditions. Native PI with correct disulfide pairing recovered under nonreducing conditions as well as total synthesized PI recovered under reducing conditions were quantified and analyzed in B (n = 3). The relative recovery of native PI in the cells expressing PI-WT was set to 100%. C: 293 cells transfected with plasmids encoding PI-WT, C(A7)Y, Y(B16)A, Y(B16)D, or Y(B16)P were metabolic labeled for 30 min followed by 0 or 2 h chase as indicated. The chase media (M) were collected and cells (C) were lysed. Biosynthesis and secretion of mutant PIs were analyzed by IP with anti-insulin followed by 4–12% NuPage. D: The results from C (n = 3) were quantified; the secretion efficiency of PI-WT and mutants was calculated, and that of PI-WT was set to 100%. E: Min6 cells were transiently transfected to express Myc-tagged PI-WT or mutants as indicated. Misfolded DLPCs in the cell lysates analyzed by 4–12% NuPage under both reducing and nonreducing conditions (left and middle panels). The media were incubated overnight and were used to analyzed secretion efficiency of PI-WT and mutants (right panel). *Refers to nonspecific bands. F: In three independent experiments shown in E, the disulfide-linked dimer and trimer under nonreducing condition and total PI-WT or mutants under reducing condition were quantified. The ratios (dimer + trimer/total PI) were calculated and that of PI-WT was set to 1. G: The secretion efficiency (media/cell lysates) of PI-WT and mutants from three independent experiments shown in E was calculated, and that of the PI-WT was set to 100%. ns, not significant.