Title: Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization
Abstract: Article3 February 2005free access Structure of palmitoylated BET3: insights into TRAPP complex assembly and membrane localization Andrew P Turnbull Andrew P Turnbull Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Protein Structure Factory, c/o BESSY GmbH, Berlin, Germany Search for more papers by this author Daniel Kümmel Daniel Kümmel Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Search for more papers by this author Bianka Prinz Bianka Prinz Institut für Biotechnologie, FG Mikrobiologie und Genetik, Technische Universität Berlin, Berlin, Germany Protein Structure Factory, Heubnerweg, Berlin, Germany Search for more papers by this author Caterina Holz Caterina Holz Institut für Biotechnologie, FG Mikrobiologie und Genetik, Technische Universität Berlin, Berlin, Germany Protein Structure Factory, Heubnerweg, Berlin, Germany Search for more papers by this author Jeffrey Schultchen Jeffrey Schultchen Institut für Biotechnologie, FG Mikrobiologie und Genetik, Technische Universität Berlin, Berlin, Germany Protein Structure Factory, Heubnerweg, Berlin, Germany Search for more papers by this author Christine Lang Christine Lang Institut für Biotechnologie, FG Mikrobiologie und Genetik, Technische Universität Berlin, Berlin, Germany Protein Structure Factory, Heubnerweg, Berlin, Germany Search for more papers by this author Frank H Niesen Frank H Niesen Protein Structure Factory, Heubnerweg, Berlin, Germany Institut für Medizinische Physik und Biophysik, Charité Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Klaus-Peter Hofmann Klaus-Peter Hofmann Institut für Medizinische Physik und Biophysik, Charité Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Heinrich Delbrück Heinrich Delbrück Protein Structure Factory, Heubnerweg, Berlin, Germany Institut für Chemie/Kristallographie, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Joachim Behlke Joachim Behlke Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Search for more papers by this author Eva-Christina Müller Eva-Christina Müller Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Search for more papers by this author Ernst Jarosch Ernst Jarosch Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Search for more papers by this author Thomas Sommer Thomas Sommer Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Search for more papers by this author Udo Heinemann Corresponding Author Udo Heinemann Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Institut für Chemie/Kristallographie, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Andrew P Turnbull Andrew P Turnbull Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Protein Structure Factory, c/o BESSY GmbH, Berlin, Germany Search for more papers by this author Daniel Kümmel Daniel Kümmel Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Search for more papers by this author Bianka Prinz Bianka Prinz Institut für Biotechnologie, FG Mikrobiologie und Genetik, Technische Universität Berlin, Berlin, Germany Protein Structure Factory, Heubnerweg, Berlin, Germany Search for more papers by this author Caterina Holz Caterina Holz Institut für Biotechnologie, FG Mikrobiologie und Genetik, Technische Universität Berlin, Berlin, Germany Protein Structure Factory, Heubnerweg, Berlin, Germany Search for more papers by this author Jeffrey Schultchen Jeffrey Schultchen Institut für Biotechnologie, FG Mikrobiologie und Genetik, Technische Universität Berlin, Berlin, Germany Protein Structure Factory, Heubnerweg, Berlin, Germany Search for more papers by this author Christine Lang Christine Lang Institut für Biotechnologie, FG Mikrobiologie und Genetik, Technische Universität Berlin, Berlin, Germany Protein Structure Factory, Heubnerweg, Berlin, Germany Search for more papers by this author Frank H Niesen Frank H Niesen Protein Structure Factory, Heubnerweg, Berlin, Germany Institut für Medizinische Physik und Biophysik, Charité Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Klaus-Peter Hofmann Klaus-Peter Hofmann Institut für Medizinische Physik und Biophysik, Charité Universitätsmedizin Berlin, Berlin, Germany Search for more papers by this author Heinrich Delbrück Heinrich Delbrück Protein Structure Factory, Heubnerweg, Berlin, Germany Institut für Chemie/Kristallographie, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Joachim Behlke Joachim Behlke Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Search for more papers by this author Eva-Christina Müller Eva-Christina Müller Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Search for more papers by this author Ernst Jarosch Ernst Jarosch Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Search for more papers by this author Thomas Sommer Thomas Sommer Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Search for more papers by this author Udo Heinemann Corresponding Author Udo Heinemann Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany Institut für Chemie/Kristallographie, Freie Universität Berlin, Berlin, Germany Search for more papers by this author Author Information Andrew P Turnbull1,2, Daniel Kümmel1, Bianka Prinz3,4, Caterina Holz3,4, Jeffrey Schultchen3,4, Christine Lang3,4, Frank H Niesen4,5, Klaus-Peter Hofmann5, Heinrich Delbrück4,6, Joachim Behlke1, Eva-Christina Müller1, Ernst Jarosch1, Thomas Sommer1 and Udo Heinemann 1,6 1Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Germany 2Protein Structure Factory, c/o BESSY GmbH, Berlin, Germany 3Institut für Biotechnologie, FG Mikrobiologie und Genetik, Technische Universität Berlin, Berlin, Germany 4Protein Structure Factory, Heubnerweg, Berlin, Germany 5Institut für Medizinische Physik und Biophysik, Charité Universitätsmedizin Berlin, Berlin, Germany 6Institut für Chemie/Kristallographie, Freie Universität Berlin, Berlin, Germany *Corresponding author. Max-Delbrück-Centrum für Molekulare Medizin, Forschungsgruppe Kristallographie, Robert-Rössle-Straße 10, 13092 Berlin, Germany. Tel.: +49 30 9406 3420; Fax: +49 30 9406 2548; E-mail: [email protected] The EMBO Journal (2005)24:875-884https://doi.org/10.1038/sj.emboj.7600565 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info BET3 is a component of TRAPP, a complex involved in the tethering of transport vesicles to the cis-Golgi membrane. The crystal structure of human BET3 has been determined to 1.55-Å resolution. BET3 adopts an α/β-plait fold and forms dimers in the crystal and in solution, which predetermines the architecture of TRAPP where subunits are present in equimolar stoichiometry. A hydrophobic pocket within BET3 buries a palmitate bound through a thioester linkage to cysteine 68. BET3 and yeast Bet3p are palmitoylated in recombinant yeast cells, the mutant proteins BET3 C68S and Bet3p C80S remain unmodified. Both BET3 and BET3 C68S are found in membrane and cytosolic fractions of these cells; in membrane extractions, they behave like tightly membrane-associated proteins. In a deletion strain, both Bet3p and Bet3p C80S rescue cell viability. Thus, palmitoylation is neither required for viability nor sufficient for membrane association of BET3, which may depend on protein–protein contacts within TRAPP or additional, yet unidentified modifications of BET3. A conformational change may facilitate palmitoyl extrusion from BET3 and allow the fatty acid chain to engage in intermolecular hydrophobic interactions. Introduction Intracellular targeting and transport of proteins in eukaryotic cells depend on a variety of proteins and protein complexes involved in the coating, budding, release, uncoating, tethering and fusion of vesicles. Vesicle budding commonly relies on formation of a protein coat around the bud and is initiated by the binding of a GTPase on the exit membrane. Subsequently, the GTPase interacts with a GTPase-activating protein, leading to GTP hydrolysis and the recruitment of coat-forming proteins to the vesicle bud. These coat-forming proteins interact with proteins on the membrane, initially leading to polymerization and stabilization of the bud and subsequently to dissection of the vesicle from the membrane (Barlowe, 1995; Springer et al, 1999). Vesicle fusion to the target compartments is triggered by membrane proteins termed soluble N-ethyl-sensitive factor (NSF) attachment protein (SNAP) receptors or SNAREs that have to be located on both the vesicle (as v-SNARE) and the target membranes (as t-SNARE) and which interact with a number of soluble factors. In addition to the interaction with SNAPs, binding of an AAA ATPase, NSF and membrane-specific multi-protein complexes to the vesicle is essential for tethering and fusion (reviewed by Hay and Scheller, 1997; Nichols and Pelham, 1998; Jahn et al, 2003; Bonifacino and Glick, 2004). The attachment of vesicles to their target membrane is mediated by a Rab GTPase and tethering factors. These tethers are multi-component complexes which are transport process-specific (Lowe, 2000; Whyte and Munro, 2002). For example, a complex called exocyst is involved in the tethering of exocytotic vesicles to the plasma membrane (TerBush and Novick, 1995). Along with the conserved oligomeric Golgi (COG) and Golgi-associated retrograde protein (GARP) complexes, the exocyst has been classified as a quadrefoil tethering complex (Whyte and Munro, 2002). The TRAPP complexes, involved in vesicle transport to the Golgi, are unrelated to the quadrefoil complexes. TRAPP has been reported to act as a guanine nucleotide exchange factor for the Rab GTPase Ypt1p (Jones et al, 2000; Wang et al, 2000). The TRAPP I complex, necessary for tethering ER-derived vesicles to the Golgi, comprises approximately equimolar amounts of seven proteins (Bet3p, Bet5p, Trs20p, Trs23p, Trs31p, Trs33p and Trs85p; yeast nomenclature) whose sequences are highly conserved from yeast to plants and mammals (Sacher et al, 2000, 2001; Sacher and Ferro-Novick, 2001). The TRAPP II complex that guides vesicles inside the Golgi comprises TRAPP I and three additional proteins: Trs65p, Trs120p and Trs130p. The individual deletion of many TRAPP complex components is lethal (Sacher et al, 2000), highlighting their importance for vesicle transport and, possibly, other cellular processes. A tagged human BET3 has been used to purify a human TRAPP complex and to identify its subunits (Gavin et al, 2002). BET3 was identified by a synthetic lethal screen as a yeast gene with a role in targeting and fusion of ER-derived vesicles to the Golgi membrane (Rossi et al, 1995). The bet3-1 mutant showed a defect in secreting invertase, α-factor and carboxypeptidase Y, leading to accumulation of these proteins at the ER-to-Golgi stage of transport prior to SNARE complex formation. Sequence analysis identified two novel motifs, [44]LX2#GX2#GX2LXE[57] and [133]G#2XGXL[139] (where X represents any amino acid and # corresponds to a hydrophobic residue; human BET3 numbering), conserved between Bet3p and two other TRAPP subunits: Trs31p and Trs33p (Sacher et al, 2000). The bet3-1 mutant, in which the TRAPP complex fails to form, involves alteration of the second conserved glycine residue (G52) in the first motif, underscoring the importance of this motif in Bet3p function (Figure 1). Figure 1.Structure-based multiple sequence alignment of BET3 homologs (Gönczy et al, 2000; Glöckner et al, 2002; Klein et al, 2002; Mammalian Gene Collection (MGC) Program Team, 2002; Wood et al, 2002). The secondary structure assignment for human BET3 is represented by cylinders (α-helix) and arrows (β-strand) above the aligned sequences. The triangles below the sequence alignment denote the extent of the assigned human BET3 structure described in this paper (residues 13–171 with respect to the wild-type protein sequence). Fully conserved residues are highlighted in red and additional, highly conserved residues (identical in at least six out of eight aligned sequences) are shaded green. Residues marked by an asterisk below the aligned sequences denote the 27 residues lining the hydrophobic pocket with any atom within 6 Å of the palmitoyl group (including L18 from the adjacent molecule in the BET3 dimer). A yellow arrow points towards the residue C68. The multiple sequence alignment was prepared using CLUSTAL (Higgins et al, 1992). Download figure Download PowerPoint BET3 is a central subunit of TRAPP that has been used to precipitate the intact tethering complex both from yeast and from human cells (Sacher et al, 1998; Gavin et al, 2002). As the first step towards a structural characterization of TRAPP, here we present the crystal structure of human BET3, a 20-kDa protein comprising 180 amino acids with 54% identity to its yeast ortholog (Sacher et al, 1998). We show BET3 to be dimeric in the crystal and in solution and identify a covalent modification with a palmitate deeply buried within a hydrophobic channel of the protein. These findings have important implications for the architecture of the TRAPP I complex and its mode of Golgi membrane attachment. Results and discussion Structure analysis and quality of the model For affinity purification of BET3, the protein was labeled with a His6 and a StrepII tag, fused to its N- and C-termini, respectively (corresponding to a construct comprising 200 amino acids). The fusion protein had a calculated molecular mass (Mr) of 22 582 kDa, but eluted from the size-exclusion chromatography column at an Mr of approximately 42 kDa (data not shown). The structure of BET3 was determined by the single-wavelength anomalous diffraction (SAD) technique and refined using data to 1.55-Å resolution. In general, the quality of the electron density map is good for 151 of the expected 200 residues in the fusion protein, the exception being the loop which connects residues 114 and 121 for which the electron density is weak and segmental flexibility must therefore be assumed. Additionally, the N-terminal 21 and the C-terminal 20 residues, including the affinity tags, are not visible in the electron density map and the structure described here is 12 and nine residues, respectively, shorter than the wild-type protein. The side chains of eight residues (K13, E35, E57, K84, F115, L118, N121 and R170) have weak density associated with them and a further seven side chains (K32, K46, R62, R67, N109, E117 and D120) are disordered and have been truncated to the last visible side chain atom in the final model. In the Ramachandran diagram, all residues of the model are located in the most favorable or additionally allowed regions (89.5 and 10.5%, respectively). BET3 structure The BET3 monomer has overall dimensions of approximately 36 × 42 × 46 Å and is constructed from four β-strands and five α-helices which harbor 16 and 39%, respectively, of the residues of the total wild-type polypeptide chain (Figure 2A). In addition, there are two segments of 310-helix (residues 120–122 and 154–156). The relationship between the elements of secondary structure and their positions in the sequence is presented in the alignment of representative BET3 homologs in Figure 1. Figure 2.Stereo diagrams of human BET3. (A) Schematic representation of the BET3 monomer with the helices and strands colored red and cyan, respectively, and labeled. The palmitate molecule covalently linked to residue C68 through a thioester bond is shown in atom colors (carbon, grey; oxygen, red; sulfur, yellow). (B) Cα trace with every tenth residue dotted and numbered. The pattern of sequence conservation across members of the BET3 family is also illustrated using the coloring scheme outlined in Figure 1. Unless otherwise stated, molecular drawings were prepared using CHIMERA (Huang et al, 1996). Download figure Download PowerPoint The secondary structural elements of BET3 are arranged in an α/β-plait topology constructed by a twisted, antiparallel, four-stranded β-sheet on one side, with the helices forming the other side of the structural motif. The pattern of sequence conservation between BET3 homologs indicates that both identical and highly conserved residues are primarily associated with the α-helical face of the molecule (Figure 2B), implying that the β-sheet simply acts as a structural scaffold. Additionally, residues 110–119 in the flexible loop connecting βb to α5 are highly conserved, which suggests that they most likely represent a binding motif for one of the other components of the TRAPP complex. Database searches using VAST (Madej et al, 1995; Gibrat et al, 1996) and DALI (Holm and Sander, 1993) reveal only limited topological similarities between BET3 and other proteins in the Protein Data Bank (PDB). Specifically, these relate to overlaps of elements of the α/β-plait fold with members of the (α+β) class of proteins. Overall, these similarities do not suggest that the PDB contains any protein with a fold significantly similar to BET3. The recently released structures of mouse BET3 (PDB entries 1VPG, 1WC8, 1WC9) show this protein to share a closely similar fold with human BET3. BET3 dimer BET3 forms a dimer around the crystallographic two-fold axis, primarily involving interactions between α1 and, to a lesser extent, α2, α4 and the N-terminal segment of α5 (Figure 3A). Analysis of the molecular surface buried between these subunits shows that the overall accessible surface area (Lee and Richards, 1971) of the BET3 monomer is approximately 9000 Å2 and that 18% (∼1500 Å2) of the subunit surface area is buried in the crystallographic dimer. Furthermore, the residues located at the dimer interface are highly conserved among the sequences of BET3 homologs (see Figures 1 and 2B). A dimeric arrangement is consistent with the biophysical data reported in this paper and with previous studies, which suggested that there are at least two copies of Bet3p and the other protein subunits in the fully assembled TRAPP complex (Sacher et al, 2000). Figure 3.BET3 dimer in the crystal and in solution. (A) Cα trace of the BET3 dimer viewed down the crystallographic two-fold axis. Secondary structural elements interacting at the dimer interface are labeled. The two subunits are shown in different colors. (B) Concentration dependence of the relative molecular mass (Mr) of BET3 in aqueous buffer from analytical ultracentrifugation, confirming a dimeric quaternary structure. The dotted line represents a monomer–dimer equilibrium fit with a Kd of 448 nM. Download figure Download PowerPoint The dimeric structure of BET3 adopted in the crystal has been additionally confirmed by analytical ultracentrifugation (Figure 3B). The molecular mass (Mr) obtained from sedimentation equilibrium experiments ranged between 43.2 kDa at a protein concentration of 0.62 g l−1 and 41.2 kDa at 0.17 g l−1. These values are somewhat lower than the expected value for the dimeric protein, and the positive concentration dependence of Mr is typical for an equilibrium between monomers and dimers. Based on the partial concentrations of both species at all Mr values, an equilibrium dissociation constant (Kd) of 448±34 nM was calculated. Given this affinity, we cannot be certain that in cells BET3 is always present as a dimer, but a dimeric structure seems very likely in compartments enriched in BET3. BET3 palmitoylation A striking feature of the BET3 structure is the presence of a hydrophobic pocket within the core of the α-helical face that is approximately 20 Å in length and is formed by the C-terminal end of α2, the loop connecting α2 to α3 and helices α3 to α5 (see Figure 2A). Additionally, within the dimer, the N-terminal end of α1 blocks the base of the pocket adjacent to the subunit interface in the symmetry-related molecule (see Figure 3A). An elongated electron density feature spans the pocket (Figure 4A) in the structures of both the wild-type and the selenomethionine-incorporated protein (data not shown), and is therefore unlikely to be an artifact of crystallization. The shape of the electron density is consistent with it being a palmitate molecule covalently attached to the protein through a thioester linkage to the fully conserved residue C68 (C-S separation 1.9 Å), corresponding to C80 in Bet3p. There are 27 highly conserved and predominantly hydrophobic residues that line the pocket with any atom within 6 Å of the S-palmitoyl group (Figure 4B). In all, 20 are identical in at least six of the eight aligned BET3 sequences, whereas conservative substitutions are seen at the remaining seven positions: V65 (L/I/W), G66 (P/S), H69 (E/N/S/A), I78 (L/V), A82 (G), Y86 (F), and L135 (I/M) (see Figure 1, human BET3 sequence numbering). The recently published structure of mouse BET3 (Kim et al, 2005) also shows a fatty acid moiety occupying this site. Palmitate is the most commonly found S-linked fatty acid (Dunphy and Linder, 1998), implying that palmitoylation of BET3 is a natural modification likely to play a functional role. The occlusion of the fatty acid chain inside a hydrophobic crevice of the protein explains why BET3 could be purified as a soluble protein from recombinant yeast cells. Figure 4.Covalent modification of BET3 with palmitate. (A) A portion of the (2m∣Fobs∣−D∣Fcalc∣)αcalc electron density map centered on the palmitoyl chain and contoured at 1.2σ (prepared using PyMOL; DeLano, 2003). The separation between the C1-atom position of the palmitoyl group and the side chain Sγ of C68 is approximately 1.9 Å, clearly indicating that a thioester bond has formed between these two entities. (B) The side chains of the 27 residues lining the hydrophobic pocket with at least one atom residing within 6 Å of the palmitate molecule are shown using the same coloring scheme as outlined in Figure 1. In all, 20 of these residues are identical in at least six of the eight aligned BET3 sequences, whereas conservative substitutions are seen at the remaining seven positions: V65 (L/I/W), G66 (P/S), H69 (E/N/S/A), I78 (L/V), A82 (G), Y86 (F), L135 (I/M). (C) Metabolic labeling with [3H]palmitate. AH22ura3 yeast strain with and without BET3 plasmids was grown in the presence of [3H]palmitic acid. BET3 and Bet3p were purified and samples were analyzed by SDS–PAGE and fluorography. Download figure Download PowerPoint The palmitoylation of BET3 in vivo was shown with metabolic labeling of yeast cells expressing BET3, Bet3p and the corresponding cysteine-to-serine mutants (BET3 C68S, Bet3p C80S). Wild-type BET3 and Bet3p purified from these cultures were palmitoylated as shown by fluorography, whereas no palmitic acid was attached to the mutant proteins (Figure 4C). Differences in intensity are due to the higher expression level of BET3 compared to Bet3p. The crystal structure of one of the other constituents of the TRAPP complex, the monomeric murine SEDL, an ortholog of yeast Trs20p, has previously been reported (Jang et al, 2002). SEDL shares a folding topology identical to the N-terminal regulatory domain of two SNAREs, yeast Ykt6p and mouse Sec22b, despite undetectable sequence homology between these proteins. This has led to the suggestion that SEDL serves regulatory and/or adaptor functions through multiple protein–protein interactions. All three proteins are involved in ER-to-Golgi vesicle transport, and the structural similarities suggest that they may interact with different partners that belong to the same family of proteins. Interestingly, the N-terminal domain of Ykt6p has been implicated in the palmitoylation of the fusion factor Vac8 (Dietrich et al, 2004). Ykt6p presents palmitoyl-CoA via its N-terminal domain to Vac8, while transfer to Vac8's SH4 domain occurs spontaneously. Furthermore, Trs20p, the SEDL ortholog in yeast, has been reported to interact with Bet3p on the basis of a genome-wide yeast two-hybrid analysis (Ito et al, 2000). This leads to the intriguing possibility that Trs20p (SEDL) exhibits the acyltransferase activity for Bet3p (BET3) palmitoylation. Possible function of BET3 palmitoylation Palmitoylation most commonly contributes to the membrane localization of proteins that would otherwise be cytoplasmic, but has also been shown to modulate protein–protein interactions (Dunphy and Linder, 1998). In theory, a single palmitoyl group should be sufficient for membrane association (Peitzsch and McLaughlin, 1993). Since previous studies have shown that TRAPP resides within a Triton X-100-resistant fraction of the Golgi (Sacher et al, 2000), we addressed the question whether BET3 palmitoylation is involved in its membrane anchoring. Preparations of cytosolic and membrane fractions from yeast cells expressing BET3 and BET3 C68S, respectively, were analyzed by immunoblotting. Wild-type and mutant BET3 were detectable both in the cytosol and the membrane (Figure 5A). By loading crude cell lysates on a sucrose cushion, it was demonstrated that BET3 as well as BET3 C68S actually are membrane associated and do not form aggregates (not shown). The membrane-bound forms of the proteins cannot be released by extraction with high salt, urea, carbonate and Triton X-100, a feature associated with tightly membrane-associated proteins (Figure 5B). These data show that BET3 is tightly bound to membranes, but that this interaction is only in part mediated through palmitoylation. We take this to suggest that the tight binding to an anchor protein or another modification is additionally required and sufficient to mediate the membrane association of BET3. Since BET3 from the soluble cytosolic pool was purified for crystallization, we cannot exclude the possibility that an additional modification is present in membrane-bound BET3. Figure 5.(A) Membrane preparations from yeast expressing BET3 and BET3 C68S. Cleared lysates were subjected to ultracentrifugation. Total cell lysate (T), cytosolic (C), and membrane (M, 10-fold concentrated) fractions were analyzed by SDS–PAGE and immunoblotting. (B) Membrane extractions of BET3 and BET3 C68S. Membranes were treated with buffer, 0.5 M NaCl, 2 M urea, 0.1 M Na2CO3, 1% Triton X-100, or 1% SDS for 30 min on ice. Samples were separated into pellet (P) and supernatant (S) fractions by centrifugation and analyzed by SDS–PAGE and immunoblotting. The integral membrane protein Sec61p and the peripheral membrane-associated proteins Cdc48p and Ubc7p served as reference. (C) Tetrad analysis of yeast Y25984 bet3 deletion strain (EUROSCARF; MAT a/α; his3Δ1/his3Δ1; leu2Δ0/leu2Δ0; lys2Δ0/LYS2; MET15/met15Δ0; ura3Δ0/ura3Δ0; YKR068c∷kanMX4/YKR068c). Sporulation was induced in cells with and without BET3 plasmids, and spores were dissected on YPD plates of individual tetrads. Download figure Download PowerPoint To address the question if palmitoylation of BET3 is essential for protein function in vivo, BET3 constructs were transformed in a yeast bet3 deletion strain, and sporulation of these transformants was induced. Loss of Bet3p is lethal and results in only two viable spores if no complementation can be achieved through the transformed constructs. In contrast to human SEDL which complements Trs20p (Gecz et al, 2003), BET3 fails to rescue haploid cells with a bet3 deletion despite the high degree of sequence conservation. Since not only Bet3p but also Bet3p C80S complement a bet3 knockout (Figure 5C), palmitoylation of Bet3p does not appear to be essential for cell viability. However, the expression of BET3 under the control of a copper promoter, even when not induced, might lead to a high intracellular level of BET3. A fine-regulatory effect of the palmitoylation on protein stabilization or protein–protein interactions may remain undetected in these cells, when they survive in the absence of a fully functional secretory system. There is some structural similarity between the palmitate-binding α-helical face of BET3 and the nonspecific lipid-transfer proteins (LTPs) from plants and mammals (Shin et al, 1995) although their complete domain folds do not match. The arrangement of three out of the four helices in barley LTP are similar in BET3 (αA, αD and αC in barley LTP are equivalent to α2, α4 and α5 in BET3; Figure 6A) and the bent helix αA in barley LTP is in a position similar to the kinked helix α2 in BET3. In barley LTP, the binding pocket can contract or expand to accommodate lipids or fatty acids of various sizes (Lerche et al, 1997; Lerche and Poulsen, 1998). These conformational changes are accompanied by a bending motion in αA and structural adjustments at the C-terminus and helix αC. Figure 6.Proposed mechanism for the extrusion of the palmitoyl group from the hydrophobic pocket. (A) Structural similarity between the α-helical face of BET3 (blue) and the nonspecific LTP from barley (yellow), here shown with bound palmitate (Lerche and Poulsen, 1998). Helices A, D and C in barley LTP adopt an arrangement similar to helices 2, 4 and 5 in BET3. The arrows point to the kink in helix α2 of BET3 and to a corresponding position within helix αA of the LTP, where a conformational change is observed when the pocket is adjusted for the binding of differently sized lipid-like ligands. (B) The BET3 monomer is shown in blue and α1′ from the other subunit of the dimer, that blocks the base of the hydrophobic pocket, is highlighted in green. The palmitoyl group is likely to play a role in membrane association but is, however, completely buried within the α-helical face of the molecule. A rotation of approximately 30° in α2 about a hin