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Exocytosis requires asymmetry in the central layer of the SNARE complex

2000; Springer Nature; Volume: 19; Issue: 22 Linguagem: Inglês

10.1093/emboj/19.22.6000

1460-2075

Rainer Ossig,

Lipid Membrane Structure and Behavior

Article15 November 2000free access Exocytosis requires asymmetry in the central layer of the SNARE complex Rainer Ossig Rainer Ossig Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Present address: Department of Physiology, University of Münster, Robert-Koch-Strasse 27a, D-48149 Münster, Germany Search for more papers by this author Hans Dieter Schmitt Hans Dieter Schmitt Department of Molecular Genetics, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Search for more papers by this author Bert de Groot Bert de Groot Department of Theoretical Molecular Biophysics Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Search for more papers by this author Dietmar Riedel Dietmar Riedel Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Search for more papers by this author Sirkka Keränen Sirkka Keränen VTT, Biotechnology, Tietotie 2, Espoo, PO Box 1500, FIN-02044 VTT, Finland Search for more papers by this author Hans Ronne Hans Ronne Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala Genetic Center, Box 7080, S-75007 Uppsala, Sweden Search for more papers by this author Helmut Grubmüller Helmut Grubmüller Department of Theoretical Molecular Biophysics Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Search for more papers by this author Reinhard Jahn Corresponding Author Reinhard Jahn Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Search for more papers by this author Rainer Ossig Rainer Ossig Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Present address: Department of Physiology, University of Münster, Robert-Koch-Strasse 27a, D-48149 Münster, Germany Search for more papers by this author Hans Dieter Schmitt Hans Dieter Schmitt Department of Molecular Genetics, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Search for more papers by this author Bert de Groot Bert de Groot Department of Theoretical Molecular Biophysics Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Search for more papers by this author Dietmar Riedel Dietmar Riedel Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Search for more papers by this author Sirkka Keränen Sirkka Keränen VTT, Biotechnology, Tietotie 2, Espoo, PO Box 1500, FIN-02044 VTT, Finland Search for more papers by this author Hans Ronne Hans Ronne Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala Genetic Center, Box 7080, S-75007 Uppsala, Sweden Search for more papers by this author Helmut Grubmüller Helmut Grubmüller Department of Theoretical Molecular Biophysics Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Search for more papers by this author Reinhard Jahn Corresponding Author Reinhard Jahn Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany Search for more papers by this author Author Information Rainer Ossig1,2, Hans Dieter Schmitt3, Bert de Groot4, Dietmar Riedel1, Sirkka Keränen5, Hans Ronne6, Helmut Grubmüller4 and Reinhard Jahn 1 1Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany 2Present address: Department of Physiology, University of Münster, Robert-Koch-Strasse 27a, D-48149 Münster, Germany 3Department of Molecular Genetics, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany 4Department of Theoretical Molecular Biophysics Group, Max-Planck-Institute for Biophysical Chemistry, D-37077 Göttingen, Germany 5VTT, Biotechnology, Tietotie 2, Espoo, PO Box 1500, FIN-02044 VTT, Finland 6Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala Genetic Center, Box 7080, S-75007 Uppsala, Sweden *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:6000-6010https://doi.org/10.1093/emboj/19.22.6000 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Assembly of SNAREs (soluble N-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells. The synaptic SNARE complex is represented by a twisted bundle of four α-helices. Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R). We have examined the functional consequences of Q–R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion. When a 3Q/1R ratio is restored by a mirror R→Q substitution in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers. Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms. We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion. Introduction SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) represent families of small and mostly membrane-bound proteins that are located on the cytoplasmic surfaces of all membranes of the secretory pathway (Götte and Fischer von Mollard, 1998; Jahn and Südhof, 1999; Mayer, 1999; Pfeffer, 1999). Membranes destined to fuse contain matching sets of SNAREs. Based on their preferred localization, SNARE proteins were classified originally as either v-SNAREs (for SNAREs on trafficking vesicles) or t-SNAREs (for SNAREs on target membranes) (Söllner et al., 1993; Rothman, 1994). In vitro, appropriate sets of SNAREs spontaneously form tight core complexes. In vivo, complex formation probably bridges the opposing membranes (trans-complex) and ties the membranes closely together. Upon bilayer fusion, the complexes relax into the cis-orientation in which all membrane anchor domains are embedded in the same membrane (Hanson et al., 1997; Lin and Scheller, 1997; Weber et al., 1998; Ungermann et al., 1998). The complexes are then disassembled by the chaperone ATPase NSF (NEM-sensitive factor) in conjunction with cofactors termed SNAPs (soluble NSF attachment proteins) (Söllner et al., 1993). The best characterized SNAREs are those involved in neuronal exocytosis. They include the vesicle protein synaptobrevin and the synaptic membrane proteins syntaxin 1 and SNAP-25. These proteins are present in a 1:1:1 stoichiometry when a core complex is formed. Deletion mutagenesis revealed that the interactions between these SNAREs are confined to a stretch of ∼60 amino acids. Syntaxin 1 and synaptobrevin each contain one such interacting region, whereas SNAP-25 contains two, one located on either side of its palmitoylated membrane anchor domain (Jahn and Südhof, 1999). Although the overall similarity between distantly related SNAREs is rather low, profile-based alignments of the interacting sequences reveal a conserved pattern of hydrophobic amino acids, in heptad repeats, referred to as the SNARE motif (Terrian and White, 1997; Weimbs et al., 1997, 1998). In the crystal structure of the neuronal complex, these amino acids form stacked leucine zipper-like layers in the core of the four-helix bundle (Sutton et al., 1998). SNARE mutations known to exhibit defects in membrane trafficking generally affect residues in the core layers, suggesting that an undisturbed layer structure is essential for function (Fasshauer et al., 1998). The central layer of the helix bundle ('0' layer) is composed of three highly polar (glutamine) and one positively charged (arginine) amino acid side chain (Sutton et al., 1998). This layer is remarkable because: (i) it is the only polar/ionic layer of the entire complex that is shielded from water by surrounding hydrophobic groups; (ii) in contrast to most other layers, it is highly asymmetric; and (iii) the amino acids contributing to this layer are almost completely conserved in the entire SNARE superfamily. For these reasons, we have recently proposed the re-classification of SNAREs into Q- and R-categories. All known t-SNAREs are Q-SNAREs, and most v-SNAREs are R-SNAREs (Fasshauer et al., 1998). The remarkable conservation of the ionic layer and also most of the hydrophobic layers strongly suggests that the overall structure determined for the neuronal SNARE complex is common to all SNARE complexes. Thus, SNARE complexes may generally consist of four-helix bundles, with three helices derived from the Q-SNAREs and one helix derived from the R-SNAREs. If formation of the helical bundle is decisive for membrane fusion, one would assume that the interactions between the amino acids in the '0' layer are essential for SNARE function. Interestingly, the arrangement of the side chains in this layer displays rotational symmetry with respect to the plane of the layer. The central position of the three arginine nitrogens in the synaptic '0' layer that participate in hydrogen bonding suggests that such a rotation is possible without incurring steric or electrostatic penalties (Sutton et al., 1998). In other words, there are no obvious structural reasons why the arginine needs to be contributed by synaptobrevin and its relatives instead of by one of the other constituents. Consequently, a switch of the arginine from synaptobrevin to syntaxin should affect neither the stability of the helical bundle nor the functioning of the SNAREs in fusion despite the high degree of conservation in these positions. Alternatively, however, the conserved glutamines and arginines may be essential for a function of the individual SNAREs unrelated to the formation of core complexes, e.g. in interactions with other proteins and/or in different conformations. In that case, an exchange of the arginine of synaptobrevin with the glutamine of one of the Q-SNAREs would be expected to impair the function of these SNAREs in membrane fusion. In the present study, we have examined how conversion of the layer '0' site of a Q-SNARE into an R and, conversely, of an R-SNARE into a Q affects SNARE function in membrane fusion. These experiments require that the endogenous SNAREs are replaced by the corresponding mutant versions. For these reasons, we used the exocytotic SNARE complex of the yeast Saccharomyces cerevisiae as a model since gene replacements can easily be performed in this organism. This SNARE complex is composed of the Q-SNAREs Sso1p/Sso2p (homologous to neuronal syntaxin) and Sec9p (homologous to neuronal SNAP-25), and the R-SNARE Snc1p/Snc2p (homologous to neuronal synaptobrevin) (Brennwald et al., 1994). Although the overall homology between the corresponding yeast and neuronal proteins is not high, the amino acids of the interacting layers are conserved to a much higher degree, with 50% being identical and most of the others being structurally very similar. Furthermore, the yeast proteins form core complexes in vitro that exhibit properties reminiscent of the neuronal proteins (Brennwald et al., 1994; Gerst, 1997; Rice et al., 1997; Katz et al., 1998; Fiebig et al., 1999), suggesting that the structures of the yeast and neuronal SNARE complexes are closely related. Moreover, structural distortions and changes in stability caused by the proposed mutations should be qualitatively similar to those of the neuronal SNARE complex. In the present work, we have, therefore, complemented the experiments with computer simulations of the mutations that were based on the crystal structure of the neuronal SNARE complex (Sutton et al., 1998). Results Effects of glutamine and arginine exchanges in SNC2 and SSO2 To examine the effects of Q/R exchanges in the '0' layer, we constructed yeast strains carrying chromosomal deletions in four genes (SNC1, SNC2, SSO1 and SSO2). SNC1 and SNC2, as well as SSO1 and SSO2, are functionally redundant genes. They encode highly homologous proteins that are required for vesicle exocytosis and cell viability under standard conditions (Aalto et al., 1993; Protopopov et al., 1993). To allow normal growth and secretion, such a deletion strain expressed alleles of SNC2 and SSO2 from centromere-based vectors under the control of the cytochrome c1 promoter. In addition to these wild-type variants, two mutant alleles were constructed: SNC2R52Q and SSO2Q228R. To analyze the functional effects of these mutations, plasmids containing epitope-tagged versions of the mutant or the corresponding wild-type sequences were introduced into the deletion strain. The resulting combinations of expressed SNC2 and SSO2 alleles, encoding an arginine or glutamine residue in the '0' layer of the SNARE proteins, respectively, are shown in Figure 1. Figure 1.Schematic illustration of the arrangement of amino acid side chains involved in the '0' layers of wild-type and mutant SNARE complexes used in this study. Local helical axes of the SNARE proteins are displayed in blue for Snc2p, red for Sso2p and green for Sec9p. Download figure Download PowerPoint Yeast transformants containing all four possible allele combinations were shown to be viable. However, expression of SSO2Q228R in the presence of wild-type SNC2, placing two arginine residues in the '0' layer, results in significantly retarded growth at 25°C and growth arrest at 37°C (R/R mutant in Figure 2). The efficiency of protein secretion was analyzed by measuring the release of invertase. In these experiments, expression of endogenous invertase was derepressed during a 1 h incubation in low glucose concentration medium. As shown in Figure 3, the growth defect is mirrored by a serious defect in invertase secretion. The R/R mutant secreted only ∼40% of total invertase at 25°C, and even less at 37°C. Although both cellular growth and secretion defects are aggravated at elevated temperature, the severe functional impairment at 25°C clearly distinguishes this phenotype from typical temperature-sensitive (Ts−) defects caused by mutations such as sso2-1 (Figure 3). To exclude the possibility that the secretion defect is confined to the invertase-containing subpopulation of vesicles (David et al., 1998), we also monitored the pattern of proteins secreted into the medium after pulse-labeling followed by precipitation and SDS–PAGE analysis (Figure 4). The pattern of secreted proteins was similar in all four strains. However, a significant reduction in the amount of media proteins was observed with the R/R mutant, which was reduced further after shifting the cells to 37°C (Figure 4). To exclude that the differences between the strains are due to differences in the expression levels of the Sso2p and Snc2p variants, the protein levels were measured in each strain by immunoblotting (not shown). In all four strains, there was no significant difference in the levels of either Snc2p or Sso2p variants. However, the expression levels under the moderate cytochrome c1 promoter were somewhat higher than expression rates of SSO and SNC genes in wild-type strains (Snc2p, ∼3-fold; Sso2p, 4- to 5-fold; at both 25 and 37°C). Figure 2.Growth curves of yeast cells expressing different sets of wild-type and mutated SNAREs. Cells of strain Δ4-2D [sso1::URA3, sso2::LEU2, snc1::URA3, snc2::ADE8] were transformed with sets of plasmids as indicated: WT Q/R (filled inverted triangles), pRS314-SSO2wt/pRS313-SNC2wt; Mut R/R (open inverted triangles), pRS314-SSO2Q228R/pRS313-SNC2wt; Mut R/Q (open circles), pRS314-SSO2Q228R/pRS313-SNC2R52Q; Mut Q/Q (filled circles), pRS314-SSO2wt/pRS313-SNC2R52Q. Cell growth was monitored by measuring the optical density at 600 nm during incubation at 25°C and following a temperature shift to 37°C (after 6 h) as described in Materials and methods. Download figure Download PowerPoint Figure 3.Invertase secretion from yeast cells (strain Δ4-2D) expressing different pairs of SSO2 and SNC2 alleles from centromere-based plasmids as indicated in Figure 2. Transformed yeast cells were derepressed for invertase and incubated at 25°C (light gray bars) or 37°C (dark gray bars). The secreted invertase activity of the different transformants is shown as a percentage of the total enzyme activity. Each bar represents the mean value of four independent experiments (mean values ± SEM). Note that the untransformed strain H603 (sso2-1) exhibits a temperature-sensitive secretion defect, showing a significant decrease in invertase release only at the non-permissive temperature. Download figure Download PowerPoint Figure 4.Secretory defects of mutants expressing the R/R combination in the '0' layer of the exocytotic SNARE complex. For a total secretion assay, yeast transformants containing SSO2 and SNC2 variants as shown in Figure 2 were radiolabeled, and secreted proteins were precipitated from the growth medium as described in Materials and methods. After separation by SDS–PAGE, radiolabeled proteins were detected by fluorography. The figure shows a representative example of several experiments. We also determined incorporation of radioactivity into total cell protein that was roughly comparable in all strains. Although the pattern of secreted proteins is similar for all strains, the R/R mutant obviously displays a reduced secretion efficiency exhibited by the reduced amount of media proteins. Download figure Download PowerPoint The data described so far demonstrate that the replacement of the glutamine with an arginine in the Q-SNARE Sso2p, resulting in a 2Q/2R '0' layer, severely disrupts the function of the SNARE complex. We then examined whether normal function can be restored when the reciprocal R→Q exchange is introduced in Snc2p. This complementary mutation (SNC2R52Q) re-establishes the 3Q/1R ratio of the original complex but effectively 'rotates' the arginine in the '0' layer from the Snc2p helix to the neighboring Sso2p helix. Indeed, we found that both growth rate and protein secretion of this strain were indistinguishable from those of the strain expressing only wild-type proteins, even at the critical temperature of 37°C (Figures 2, 3 and 4). Moreover, the defects caused by the SSO2Q228R allele could also be rescued by expressing SNC2R52Q in yeast cells still harboring the chromosomal SNC wild-type genes (not shown). This result illustrates that the presence of two arginine residues in the '0' layer severely disrupts SNARE function; nevertheless, it does not induce the formation of a 'dead-end' complex, which would result in a dominant-negative phenotype. Together, these findings show that the arginine can be swapped to a different helix in the '0' layer without affecting the function of the complex. Yeast cells containing the Q/Q combination (see Figure 1) expressed SNC2R52Q in the presence of wild-type Q-SNAREs, leading to only glutamines in the '0' layer of the SNARE complex. This investigation was encouraged further by reports suggesting the involvement of SNARE complexes that contain only Q-SNAREs in certain intracellular fusion steps (Patel et al., 1998; Nichols et al., 1997; Rabouille et al., 1998). Surprisingly, both growth rate and protein secretion of these cells (Figures 2, 3 and 4) were almost indistinguishable from those of the strain expressing the wild-type genes. The slight differences when compared with the wild-type and the R/Q strain [evident, for instance, by the morphological appearance (see below) and by the slightly reduced growth rate (Figure 2)] were difficult to establish unequivocally. We therefore investigated whether these minor defects become more evident when analyzed in conjunction with defects in other layers of the SNARE complex. To verify whether SNC2R52Q is functionally different from its wild-type counterpart, we first tested its ability to suppress other secretion defects. For this purpose, we selected a sec9-4 mutation strain containing a glycine to aspartic acid substitution in the −3 layer of the SNAP-25 homolog Sec9p (Brennwald et al., 1994) and a sso2-1 strain that carries an arginine to lysine substitution in the −8 layer. As already observed previously (Couve and Gerst, 1994; Gerst, 1997), the temperature-sensitive growth defects of these two mutant strains could be partially compensated by overexpression of wild-type SNC2. In addition, we used a strain expressing a Ts− mutation in SEC1. This strain is also rescued at restrictive temperature by overexpression of wild-type SNC2 (Couve and Gerst, 1994). Unlike SSO2 and SEC9, SEC1 encodes an essential SNARE-interacting protein that is not part of the SNARE core complex. Thus, the sec1-1 mutation can serve as control for a functional rescue that does not involve SNARE interactions in the core complex. We observed that, unlike overexpression of wild-type SNC2, high level expression of SNC2R52Q was unable to restore growth of both sso2-1 and sec9-4 cells at the restrictive temperature of 34°C. In contrast, expression of both alleles was found to be equally effective in rescuing sec1-1 yeast cells (Figure 5, upper panel). More importantly, a synergistic negative effect of SNC2R52Q in conjunction with sso2-1 was observed at temperatures >30°C, i.e. a temperature range that still permits cellular growth of the sso2-1 strain. Overexpression of SNC2R52Q strongly inhibited cellular growth even in the presence of wild-type SNC genes (Figure 5, lower panel). With sec9-4, such a synergistic negative effect could not be observed. However, by using two different approaches, we were unable to obtain sec9-4 mutant cells that express the SNC2R52Q allele in the absence of wild-type SNC genes, strongly suggesting that SNC2R52Q is synthetically lethal in combination with the sec9-4 mutation. Thus, SNARE complexes with 4Q in the '0' layer are capable of mediating membrane fusion at an efficiency close to that of 3Q/1R complexes. However, they do contain a structural defect that becomes more evident when combined with defects in other layers of the four-helix bundle. Figure 5.Genetic interaction between two SNC2 alleles and three mutations affecting the function of the exocytic SNARE complex in yeast. Upper panel: the two different SNC2 alleles (SNC2wt and SNC2R52Q) were tested for suppression of particular conditional defects affecting vesicle fusion at the plasma membrane. Snc2p (wild-type) or Snc2R52Q protein was overproduced in cells carrying Ts− defects in either the SEC1 gene (sec1-1), the SEC9 gene encoding the yeast SNAP-25 homolog (sec9-4) or the SSO2 gene (sso1::URA3; sso2-1). ↑, improvement of growth relative to transformants containing the pure vector; →, no change in growth rate; ↓, inhibition of growth. Cells transformed with multicopy vector pRS323 (URA3, 2μ), pRS323-SNC2wt or pRS323-SNC2R52Q were plated on synthetic minimal medium lacking uracil. Growth was scored after 3 or 4 days of incubation at 30, 32.5 or 35°C. The latter two temperatures are restrictive for growth of sec1-1, sec9-4 and sso2-1 mutants. Note that in contrast to the wild-type allele, the SNC2R52Q allele was unable to suppress the growth defect of sec9-4 mutants and even aggravated the growth defect caused by the sso2-1 mutation. However, overexpression of SNC2R52Q did sustain the growth of sec1-1 cells as efficiently as wild-type SNC2. Lower panel: sso2-1 mutants incubated at 32.5°C for 3 days. The mutants contained either pRS323 without insert (lower third of the plate), pRS323-SNC2 (upper left) or pRS323-SNC2R52Q (upper right). sso2-1 yeast transformants overexpressing SNC2R52Q showed a synergistic negative effect. Download figure Download PowerPoint For further characterization of our mutants, we analyzed the morphology of the four strains by light and electron microscopy. When monitored by differential interference contrast (DIC) microscopy, the R/R mutant had a clearly abnormal appearance (Figure 6). Many cells were unusually large, and cell groups that apparently had not separated after division were evident. Furthermore, cells of this strain lack the vacuolar structures that were visible as indentations within wild-type cells. At the electron microscopic level, the R/R mutant was characterized by an accumulation of numerous vesicles with diameters of 80–100 nm that correspond to the size of post-Golgi vesicles (Figure 6). This phenotype is indicative of a defect in the final step of the secretory pathway. In contrast, the R/Q 'swap' mutant was morphologically indistinguishable from the strain expressing the wild-type combination. The Q/Q mutant also looked normal except that the cell size distribution was slightly more heterogeneous and the vacuole often had a more fragmented appearance. Figure 6.Morphology of cells expressing different '0' layer Q/R combinations analyzed by light microscopy (A, B, C and D) and electron microscopy (E, F, H and I, bar = 1 μm; G, bar = 370 nm; J, bar = 100 nm). Transformants of strain Δ4-2D were used as follows: (A), (E) and (J) WT Q/R, pRS314-SSO2wt/pRS313-SNC2wt; (B), (F), (G) and (K) Mut R/R, pRS314-SSO2Q228R/pRS313-SNC2wt; (C) and (H) Mut R/Q, pRS314-SSO2Q228R/pRS313-SNC2R52Q; (D) and (I) Mut Q/Q, pRS314-SSO2wt/pRS313-SNC2R52Q. Cells were grown and treated at 25°C before light microscopic inspection with DIC or incubated at 37°C for 45 min before fixation for electron microscopy. (J and K) Higher magnifications of the WT Q/R and Mut R/R strains that were obtained from spheroblasts using a different fixation and staining protocol. Note the aberrant cell shape and the numerous vesicles that are visible in the mutant containing two arginines in the '0' layer of the exocytotic SNARE complex. Download figure Download PowerPoint We also performed immunofluorescence microscopy in order to localize the Snc2p and Sso2p variants in all four strains. As shown in Figure 7, the plasma membrane localization of the Sso2Q228R protein was found to be indistinguishable from that of wild-type Sso2p regardless of whether the R or Q variant of Snc2p was expressed. These observations suggest that the defect in exocytosis in the R/R mutant is not due to missorting or serious degradation of the mutated protein. Interestingly, the R/R mutant showed strong and diffuse staining for Snc1p that was often enriched in the daughter cells (Figure 7). This probably reflects the accumulation of small Snc1p-containing transport vesicles as observed in the EM analysis (Figure 6). Figure 7.Indirect immunofluorescence microscopy to confirm that plasma membrane localization of the Sso2Q228R protein is indistinguishable from that of wild-type Sso2p. Yeast transformants of strain Δ4-2D containing different pairs of either wild-type (WT Q/R) or mutant (Mut R/R; Mut R/Q; Mut Q/Q) SSO2 and SNC2 alleles from centromeric plasmids (indicated in Figure 2) were fixed and prepared for immunocytochemistry. HA and c-Myc epitope-specific anibodies were used to detect the epitope-tagged SNARE proteins. Cy2-conjugated antibodies track Sso2wt or Sso2Q228R proteins (shown in green), while Cy3-conjugated antibodies labeled Snc2wt or Snc2R52Q proteins (shown in red). DAPI staining of DNA was used to localize the nucleus (blue). Download figure Download PowerPoint To exclude the possibility that other intracellular trafficking pathways are affected in any of the mutant strains (e.g. defects in vacuolar sorting as suggested by the microscopic appearance), we examined the processing of the vacuolar enzyme carboxypeptidase Y (CPY) after pulse-labeling. After translocation, this protein is core-glycosylated in the endoplasmic reticulum (p1 form), further glycosylated in the Golgi apparatus (p2 form) and proteolytically processed upon arrival in the vacuole (mature form) (Stevens et al., 1982). As shown in Figure 8, all four strains exhibited a normal pattern of CPY processing, with the R/R strain being slightly retarded (probably secondary to the overall growth defect). These findings confirm that neither endoplasmic reticulum (ER)-to-Golgi nor Golgi-to-vacuole trafficking was impaired. Figure 8.Maturation of carboxypeptidase Y was monitored in order to follow intracellular transport in strain Δ4-2D cells expressing different pairs of SSO2 and SCN2 alleles. Transformants containing SSO2 and SNC2 variants as indicated in Figure 2 were grown in synthetic medium at 25°C and pulse–chased with 35S-labeled cysteine and methionine for 5 min at 37°C. Aliquots were removed at 0, 7.5 and 15 min of chase time. Cells were separated from the medium, lysed, and lysates and medium samples were subjected to immunoprecipitation with CPY-specific antibodies. The positions of the ER precursor (p1), the Golgi-modified precursor (p2) and the mature vacuolar enzyme (m) are indicated. Download figure Download PowerPoint Calculation of free energy changes for Q/R substitutions in neuronal SNAREs using molecular dynamics simulations The phenotypes described above suggest that an interference with the side chain interactions of the '0' layer affects SNARE function in membrane fusion. However, it remains unclear to what extent the amino acid substitutions affect the structure of the '0' layer region and the overall stability of the complex. We have therefore performed five molecular dynamics free energy calc