Figure 1.

Translocation defect caused by R6C mutation occurs only in the GFP-tagged preproinsulin shorter than 160 amino acids. A, full-length and truncated GFP-tagged preproinsulins with different lengths ranging from 348 to 110 amino acids (aa). The signal peptide (SP, red), insulin B-chain (light blue), C-peptide (yellow), insulin A-chain (blue), and the insertion site of GFP (green) are indicated. Authentic preproinsulin without tag is shown in the lower panel. B, 293T cells were transfected with plasmids encoding different lengths of preproteins shown in A with wildtype (WT) or R6C signal sequence. At 40 h post-transfection, the cells were labeled with [³⁵S]Met/Cys for 10 min without chase. The newly synthesized preproteins were immunoprecipitated with anti-insulin antibody. Uncleaved preproteins (P) and cleaved mature proteins (M) are indicated. The SP cleavage site mutation preproCpepGFP-A24D was used as a control of full-length signal-uncleaved preproCpepGFP. < indicates nonspecific bands. C, newly synthesized preproteins shown in B from as least three independent experiments were quantified using ImageJ. The percentages of signal-uncleaved preproteins in the corresponding total newly synthesized wildtype (WT) and R6C mutant preproteins were calculated and are shown as mean ± S.D. *, p < 0.05 compared with WT. D, increases of percentages of signal-uncleaved preproteins of the mutants minus that of the WT were calculated and shown as mean ± S.D. *, p < 0.05 comparing to mutant preprotein with a length of 200 residues.