Behavior in the Eukaryotic Secretory Pathway of Insulin-containing Fusion Proteins and Single-chain Insulins Bearing Various B-chain Mutations

Abstract

In the secretory pathway, endoproteolytic cleavage of the insulin precursor protein promotes a change in the biophysical properties of the processed insulin product, and this may be relevant for its intracellular trafficking. We have now studied several independent point mutants contained within the insulin B-chain, S9D, H10D, V12E (called B9D, B10D, and B12E), as well as the double point mutant P28K,K29P (B28K,B29P), that have been reported to inhibit insulin oligomerization. In yeast cells, the unprocessed precursor of each of these mutants is secreted, whereas >90% of the endoproteolytically released single-chain insulin moiety is retained intracellularly; a large portion of the B9D, B10D, and B12E single-chain insulins exhibit abnormally slow mobility upon nonreducing SDS-PAGE, despite normal mobility upon reducing SDS-PAGE. Although no free thiols can be detected, each of these mutants exhibits increased disulfide accessibility to dithiothreitol. After dithiothreitol treatment, a portion of the molecules can reoxidize to a form more compact than the original single-chain insulin mutants formed in vivo (indicating initial disulfide mispairing). Disulfide mispairing of a fraction of B9D, B10D, and B12E mutants also occurs in the context of single-chain insulin and even in authentic proinsulin expressed within the secretory pathway of mammalian cells. We conclude that analyses of the intracellular trafficking of certain oligomerization-defective insulin mutants is complicated by the formation of disulfide isomers in the secretory pathway.

Role of the Connecting Peptide in Insulin Biosynthesis

“…Nevertheless, we found surprising the recent result that certain B-chain point mutations, including ones known to provide thermodynamic stability to insulin that has been folded in vitro (such a substitution of insulin HisB10 by Asp), induce insulin disulfide mispairing within the secretory pathway in vivo (12). In this study, we have primarily used analysis by nonreducing SDS-PAGE to assess insulin disulfide maturation, which is generally recognized to occur within the endoplasmic reticulum (22), focusing primarily on the length of the linker peptide contained within SCIs.…”

“…1). These data are highly reminiscent of disulfide mispairing observed upon introduction of Bchain point mutations in SCIs or proinsulin (12). Although not exhaustively explored in this study, when the linker was short, small sequence changes did affect the nonreducing SDS-PAGE mobility of the predominant isoform of insulin.…”

“…(As described previously, the gel solutions were not routinely de-gassed before use (12); however, after the experiments for this paper were completed we discovered that a small but variable degree of thiol reoxidation can occur in vitro for our samples during the running of the gel itself, and this can be effectively decreased by de-gassing the gel solutions.) Insulin gels were fixed initially in 20% trichloroacetic acid without alcohol, then in 12.5% trichloroacetic acid plus 50% methanol, then incubated briefly with water, and finally either phosphorimaged or incubated with 1 M sodium salicylate for 20 min and exposed to XAR film at Ϫ70°C.…”

A Role For Lte1p (a Low Temperature Essential Protein Involved in Mitosis) in Proprotein Processing in the Yeast Secretory Pathway

“…A genetic screen has previously been performed with yeast mutagenized by random gene disruptions (by insertion of lacZ/ LEU2) to identify genetically engineered cells exhibiting enhanced immunoreactive insulin secretion (eis, 2 derived from an integrated GAL1 promoter-driven insulin-containing fusion protein called ICFP, Ref. 1).…”

“…The ICFP is comprised of the leader and propeptide of the yeast alpha-mating factor contiguous with a single chain insulin, separated by a Kex2p cleavage site. Cleavage at the Kex2p-processing site causes insulin to be delivered ultimately to the vacuole in a manner that requires specific insulin structural features (2). From 90,000 transformants that were originally screened, six independent gene disruptions were identified.…”