In eukaryotic cells, secretion is achieved by vesicular transport. Fusion of such vesicles with the correct target compartment relies on SNARE proteins on both vesicle (v-SNARE) and the target membranes (t-SNARE). At present it is not clear how v-SNAREs are incorporated into transport vesicles. Here, we show that binding of ADP-ribosylation factor (ARF)–GTPase-activating protein (GAP) to ER-Golgi v-SNAREs is an essential step for recruitment of Arf1p and coatomer, proteins that together form the COPI coat. ARF-GAP acts catalytically to recruit COPI components. Inclusion of v-SNAREs into COPI vesicles could be mediated by direct interaction with the coat. The mechanisms by which v-SNAREs interact with COPI and COPII coat proteins seem to be different and may play a key role in determining specificity in vesicle budding.

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

Insulin-like growth factor-binding protein-3 and -5 (IGFBP-3 and -5) have been shown to bind insulin-like growth factor-I and -II (IGF-I and -II) with high affinity. Previous studies have proposed that the N-terminal region of IGFBP-5 contains a hydrophobic patch between residues 49 and 74 that is required for high affinity binding. These studies were undertaken to determine if mutagenesis of several of these residues resulted in a reduction of the affinity of IGFBP-3 and -5 for IGF-I. Substitutions for residues 68, 69, 70, 73, and 74 in IG-FBP-5 (changing one charged residue, Lys 68 , to a neutral one and the four hydrophobic residues to nonhydrophobic residues) resulted in an ϳ1000-fold reduction in the affinity of IGFBP-5 for IGF-I. Substitutions for homologous residues in IGFBP-3 also resulted in a >1000-fold reduction in affinity. The physiologic consequence of this reduction was that IGFBP-3 and -5 became very weak inhibitors of IGF-I-stimulated cell migration and DNA synthesis. Likewise, the ability of IGFBP-5 to inhibit IGF-I-stimulated receptor phosphorylation was attenuated. These changes did not appear to be because of alterations in protein folding induced by mutagenesis, because the IGFBP-5 mutant was fully susceptible to proteolytic cleavage by a specific IGFBP-5 protease. In summary, residues 68, 69, 70, 73, and 74 in IGFBP-5 appear to be critical for high affinity binding to IGF-I. Homologous residues in IGFBP-3 are also required, suggesting that they form a similar binding pocket and that for both proteins these residues form an important component of the core binding site. The availability of these mutants will make it possible to determine if there are direct, non-IGF-I-dependent effects of IGFBP-3 and -5 on cellular physiologic processes in cell types that secrete IGF-I.

Sec22p is an endoplasmic reticulum (ER)-Golgi v-SNARE protein whose retrieval from the Golgi compartment to the endoplasmic reticulum (ER) is mediated by COPI vesicles. Whether Sec22p exhibits its primary role at the ER or the Golgi apparatus is still a matter of debate. To determine the role of Sec22p in intracellular transport more precisely, we performed a synthetic lethality screen. We isolated mutant yeast strains in which SEC22 gene function, which in a wild type strain background is non-essential for cell viability, has become essential. In this way a novel temperature-sensitive mutant allele, dsl1-22, of the essential gene DSL1 was obtained. The dsl1-22 mutation causes severe defects in Golgi-to-ER retrieval of ER-resident SNARE proteins and integral membrane proteins harboring a Cterminal KKXX retrieval motif, as well as of the soluble ER protein BiP/Kar2p, which utilizes the HDEL receptor, Erd2p, for its recycling to the ER. DSL1 interacts genetically with mutations that affect components of the Golgi-to-ER recycling machinery, namely sec20-1, tip20-5, and COPI-encoding genes. Furthermore, we demonstrate that Dsl1p is a peripheral membrane protein, which in vitro specifically binds to coatomer, the major component of the protein coat of COPI vesicles.Membrane-bound compartments in eukaryotic cells can fuse directly as shown for the endoplasmic reticulum (ER) 1 and mitotic Golgi fragments as well as endosomal and lysosomal compartments (homotypic fusion; see Ref. 1). However, vectorial transport between distinct compartments mainly involves small coated vesicles whose formation from the donor membrane is mediated by proteinaceous coats, either COPI, COPII, or clathrin. After uncoating, vesicles fuse selectively with an acceptor membrane (heterotypic fusion; see Ref.2). Both homotypic and heterotypic fusion events rely on specific attachment reactions to guarantee that only appropriate membranes can mix. The membrane attachment itself consists of two steps, tethering and docking, involving different sets of proteins (3, 4).Tethering factors are peripherally membrane-associated protein complexes consisting of up to 10 different subunits, which share little sequence similarity.The subsequent docking stage involves specific sets of membrane-anchored proteins, so-called SNARE proteins (SNARE is soluble NSF (for N-ethylmaleimide-sensitive fusion protein) attachment protein receptor) (5-7). SNAREs are inserted into the membrane either by a C-terminal transmembrane domain or through lipid moieties attached to C-terminal cysteine residues. In contrast to the tethering factors, all known SNARE proteins are members of either of three protein families: the syntaxins, the synaptobrevins or VAMPs, and the SNAP-25 family members. To induce membrane fusion, SNARE proteins from apposed membranes must interact in trans. The formation of a stable four-helix bundle may generate enough energy to promote mixing of the lipid bilayer (8 -10).Lipid mixing experiments using SNARE complexes reconstituted into lipid bilayer vesicl...

Dsl1p is required for Golgi-endoplasmic reticulum (ER) retrograde transport in yeast. It interacts with the ER resident protein Tip20p and with ␦-COP, a subunit of coatomer, the coat complex of COPI vesicles. To test the significance of these interactions, we mapped the different binding sites and created mutant versions of Dsl1p and ␦-COP, which are unable to bind directly to each other. Three domains were identified in Dsl1p: a Tip20p binding region within the N-terminal 200 residues, a highly acidic region in the center of Dsl1p containing crucial tryptophan residues that is required for binding to ␦-COP and essential for viability, and an evolutionarily well conserved domain at the C terminus. Most importantly, Dsl1p uses the same central acidic domain to interact not only with ␦-COP but also with ␣-COP. Strong interaction with ␣-COP requires the presence of comparable amounts of ⑀-COP or ␤-COP. Thus, the binding characteristics of Dsl1p resemble those of many accessory factors of the clathrin coat. They interact with different layers of the vesicle coat by using tandemly arranged sequence motifs, some of which have dual specificity.The structural integrity of membrane-bound organelles requires the recycling of lipids and proteins. Between Golgi and the endoplasmic reticulum (ER) 1 this retrograde transport is mediated by COPI-coated vesicles (1). The COPI coat from yeast and mammals consists of seven COP proteins (␣-, ␤Ј-, ␤-, ␥-, ␦-, ⑀-, and -COP ϭ coatomer) and the small GTPase ARF1 (2, 3). Sorting of cargo proteins to COPI vesicles can be achieved by direct binding of cargo molecules to coatomer (4 -8). This binding is often mediated by short sequence motifs displayed by cargo molecules (6, 9). The efficient sorting of cargo into COPI vesicles depends on GTP hydrolysis by ARF1 facilitated by a specific GTPase-activating proteins (10).After the uncoating, vesicles are ready to fuse with their specific target membrane (11). Fusion events rely on specific attachment reactions to guarantee that only appropriate membranes can mix. The membrane attachment itself comprises two steps, tethering and docking (12). Both steps involve different sets of proteins. Tethering factors are peripherally membrane-associated protein complexes consisting of up to 10 different subunits, which share little sequence similarity. So far, seven different tethering complexes required for at least five different transport steps were characterized in yeast (13)(14)(15)(16)(17)(18)(19)(20). In some cases, mammalian counterparts of yeast tethering factors were identified (reviewed in Ref. 21).The subsequent docking stage involves specific sets of membrane-anchored proteins, the so-called SNARE proteins (22,23). In contrast to the tethering factors, all known SNARE proteins are members of either of three protein families: the syntaxins, the synaptobrevins or VAMPs and the SNAP-25 family members. To induce membrane fusion, SNARE proteins from apposed membranes must interact in trans. The SNARE or SNARE-like proteins involved in fusion at ...