An insulin-containing fusion protein (ICFP, encoding the yeast prepro-α factor leader peptide fused via a lysine-arginine cleavage site to a single chain insulin) has been expressed in Saccharomyces cerevisiae where it is inefficiently secreted. Single gene disruptions have been identified that cause enhanced immunoreactive insulin secretion (eis). Five out of six eis mutants prove to be vacuolar protein sorting (vps)8, vps35, vps13, vps4, and vps36, which affect Golgi↔endosome trafficking. Indeed, in wild-type yeast insulin is ultimately delivered to the vacuole, whereas vps mutants secrete primarily unprocessed ICFP. Disruption of KEX2, which blocks intracellular processing to insulin, quantitatively reroutes ICFP to the cell surface, whereas loss of the Vps10p sorting receptor is without effect. Secretion of unprocessed ICFP is not based on a dominant secretion signal in the α-leader peptide. Although insulin sorting mediated by Kex2p is saturable, Kex2p functions not as a sorting receptor but as a protease: replacement of Kex2p by truncated secretory Kex2p (which travels from Golgi to cell surface) still causes endoproteolytic processing and intracellular insulin retention. Endoproteolysis promotes a change in insulin's biophysical properties. B5His residues normally participate in multimeric insulin packing; a point mutation at this position permits ICFP processing but causes the majority of processed insulin to be secreted. The data argue that multimeric assembly consequent to endoproteolytic maturation regulates insulin sorting in the secretory pathway.

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.

The penta-subunit retromer complex of yeast mediates selective retrieval of membrane proteins from the prevacuolar endosome to the trans Golgi network. In this study, we set out to generate a panel of vps35 dominantnegative mutants that disrupt retromer-mediated cargo sorting. Mapping of the mutations revealed two types of alterations leading to dominant-negative behavior of the 944-amino acid protein: (i) mutations at or near the R 98 residue or (ii) C-terminal truncations exemplified by a nonsense mutation at codon 733. Both could be suppressed by overexpression of wild-type Vps35p, suggesting that these dominant-negative mutants compete for interactions with other retromer subunits. Interestingly, Vps35-R 98 W expression destabilized Vps26p while having no effect on Vps29p stability, while Vps35-Q 733 * expression affected Vps29p stability but had no effect on Vps26p. Measurement of Vps35/Vps26 and Vps35/Vps29 pairwise associations by coimmunoprecipitation in the presence or absence of other retromer subunits indicated that the R 98 residue, which is part of a conserved PRLYL motif, is critical for Vps35p binding to Vps26p, while both R 98 and residues 733-944 are needed for efficient binding to Vps29p.

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The retromer protein complex assists in recycling selected integral membrane proteins from endosomes to the trans Golgi network. One protein subcomplex (Vps35p, Vps26p and Vps29p) combines with a second (Vps17p and Vps5p) to form a coat involved in sorting and budding of endosomal vesicles. Yeast Vps35p (yVps35) exhibits similarity to human Vps35 (hVps35), especially in a completely conserved PRLYL motif contained within an amino-terminal domain. Companion studies indicate that an R 98 W mutation in yVps35 causes defective retromer assembly in Saccharomyces cerevisiae. Herein, we find that the expression of hVps35 in yeast confers dominant-negative vacuolar proenzyme secretion and defective secretory proprotein processing. The mutant phenotype appears to be driven by hVps35 competing with endogenous yVps35, becoming incorporated into defective retromer complexes and causing proteasomal degradation of endogenous Vps26 and Vps29. Increased expression of yVps35 displaces some hVps35 to a 100 000 3 g supernatant and suppresses the dominant-negative phenotype. Remarkably, mutation of the conserved R 107 W of hVps35 displaces some of the protein to the 100 000 3 g supernatant, slows protein turnover and restores stability of Vps26p and Vps29p and completely abrogates dominant-negative trafficking behavior. We show that hVps35 coprecipitates Vps26, whereas the R 107 W mutant does not. In pancreatic beta cells, the R 107 W mutant shifts hVps35 from peripheral endosomes to a juxtanuclear compartment, affecting both mannose phosphate receptors and insulin. These data underscore importance of the Vps35 PRLYL motif in retromer subcomplex interactions and function. It has previously been shown that upon expression of an insulin-containing fusion protein in Saccharomyces cerevisiae, molecules that fail to undergo Kex2-mediated endoproteolysis are rapidly secreted, whereas the processed insulin fraction is transported to the vacuole (1). A genetic screen identified six single gene disruptions allowing enhanced immunoreactive insulin secretion because of defective Kex2-mediated endoproteolytic processing; of these, vps35 showed the strongest phenotype (1). VPS35 encodes a subunit of retromer, a coat protein complex engaged in the retrieval of recycling membrane proteins from endosomes to the trans Golgi network (2). Protein recycling is essential for protein sorting in the vacuolar/ lysosomal biogenesis pathway as well as for lumenal protein processing in both secretory and endosomal systems (3). Moreover, retromer function is essential for the development and viability of mammalian organisms (4).The core of the retromer protein complex involves a large protein subcomplex comprised of Vps35, Vps29 and Vps26 (5) along with a smaller subcomplex comprised of Vps17 and Vps5 (6). With potential contributions from ancillary proteins, these two subcomplexes assemble into a multimeric membrane coat. Of the members of the retromer complex, yeast Vps35 has been shown to directly bind to the structural information encoded in the cytosolic...