Fig. 4.

Protein interactions of nonmutant and C(A7)Y mutant versions of PI in Akita islets and INS-1 β cells expressing hProC(A7)Y-CpepGFP. (A) Islets isolated from 5-week-old male wild-type (wt) or Akita (Ak) mice were pulse-labeled with ³⁵S-labeled amino acids for 30 min. Islets without chase or chased for 2 h in the absence of secretagogues were lysed (C), and medium (M) was collected. Samples were immunoprecipitated with anti-insulin and analyzed by Tris·tricine·urea· SDS/PAGE under reduced or nonreduced conditions. The arrowhead denotes a position of aberrant mobility of nonreduced PI with improper disulfide pairing (12). The migration of fully reduced and oxidized (native) PI is indicated. Note that, under nonreduced conditions, newly synthesized PI in Akita islets is less efficiently recovered than under reduced conditions, and there is an increase of a higher-molecular-mass protein complex (open arrow) that is not detected under reduced conditions. (B) Lysates of stably transfected INS-1 cells expressing the constructs shown or pancreatic islets infected with adenovirus driving expression of hProCpepGFP (lane 5) were subjected to Western blotting with anti-GFP after SDS/PAGE under reduced or nonreduced conditions. Lane 4, uninfected control islets; lane 6, a positive control of 293T cells expressing CpepGFP. Note that hProC(A7)Y-CpepGFP is not endoproteolytically processed in β cells, indicating failure to be delivered to immature secretory granules. Also note that pancreatic islets more efficiently process hProCpepGFP to CpepGFP (>90%) than in INS-1 cells (<50%). Finally, note that most hProC(A7)Y-CpepGFP is not recovered in its normal position under nonreduced conditions (Right), with increased higher molecular mass protein complexes (open arrows) that are not detected under reduced conditions. The positions of molecular mass markers are shown on the left. (C) The same cells from B were pulse-labeled with ³⁵S-labeled amino acids for 30 min and then lysed in immunoprecipitation buffer containing 0.1% SDS. GFP-containing peptides were immunoprecipitated, and the samples were analyzed by Tris·tricine·urea· SDS/PAGE under reducing conditions to detect coimmunoprecipitation of endogenous PI. Reduced PI was identified by using direct PI immunoprecipitation from INS-1 control cells (Left).