Specific amino acids in the BAR domain allow homodimerization and prevent heterodimerization of sorting nexin 33

SNX33 (sorting nexin 33) is a homologue of the endocytic protein SNX9 and has been implicated in actin polymerization and the endocytosis of the amyloid precursor protein. SNX33 belongs to the large family of BAR (Bin/amphiphysin/Rvs) domain-containing proteins, which alter cellular protein trafficking by modulating cellular membranes and the cytoskeleton. Some BAR domains engage in homodimerization, whereas other BAR domains also mediate heterodimerization between different BAR domain-containing proteins. The molecular basis for this difference is not yet understood. Using co-immunoprecipitations we report that SNX33 forms homodimers, but not heterodimers, with other BAR domain-containing proteins, such as SNX9. Domain deletion analysis revealed that the BAR domain, but not the SH3 (Src homology 3) domain, was required for homodimerization of SNX33. Additionally, the BAR domain prevented the heterodimerization between SNX9 and SNX33, as determined by domain swap experiments. Molecular modelling of the SNX33 BAR domain structure revealed that key amino acids located at the BAR domain dimer interface of the SNX9 homodimer are not conserved in SNX33. Replacing these amino acids in SNX9 with the corresponding amino acids of SNX33 allowed the mutant SNX9 to heterodimerize with SNX33. Taken together, the present study identifies critical amino acids within the BAR domains of SNX9 and SNX33 as determinants for the specificity of BAR domain-mediated interactions and suggests that SNX9 and SNX33 have distinct molecular functions.     

Two domains within the first putative transmembrane domain of presenilin 1 differentially influence presenilinase and gamma-secretase activity

Presenilins (PS) are thought to contain the active site for presenilinase endoproteolysis of PS and gamma-secretase cleavage of substrates. The structural requirements for PS incorporation into the gamma-secretase enzyme complex, complex stability and maturation, and appropriate presenilinase and gamma-secretase activity are poorly understood. We used rescue assays to identify sequences in transmembrane domain one (TM1) of PS1 required to support presenilinase and gamma-secretase activities. Swap mutations identified an N-terminal TM1 domain that is important for gamma-secretase activity only and a C-terminal TM1 domain that is essential for both presenilinase and gamma-secretase activities. Exchange of residues 95-98 of PS1 (sw95-98) completely abolishes both activities while the familial Alzheimer's disease mutation V96F significantly inhibits both activities. Reversion of residue 96 back to valine in the sw95-98 mutant rescues PS function, identifying V96 as the critical residue in this region. The TM1 mutants do not bind to an aspartyl protease transition state analog gamma-secretase inhibitor, indicating a conformational change induced by the mutations that abrogates catalytic activity. TM1 mutant PS1 molecules retain the ability to interact with gamma-secretase substrates and gamma-secretase complex members, although Nicastrin stability is decreased by the presence of these mutants. gamma-Secretase complexes that contain V96F mutant PS1 molecules display a partial loss of function for gamma-secretase that alters the ratio of amyloid-beta peptide species produced, leading to the amyloid-beta peptide aggregation that causes familial Alzheimer's disease.     

The crystal structure of mouse Nup35 reveals atypical RNP motifs and novel homodimerization of the RRM domain

The nuclear pore complex mediates the transport of macromolecules across the nuclear envelope (NE). The vertebrate nuclear pore protein Nup35, the ortholog of Saccharomyces cerevisiae Nup53p, is suggested to interact with the NE membrane and to be required for nuclear morphology. The highly conserved region between vertebrate Nup35 and yeast Nup53p is predicted to contain an RNA-recognition motif (RRM) domain. Due to its low level of sequence homology with other RRM domains, the RNP1 and RNP2 motifs have not been identified in its primary structure. In the present study, we solved the crystal structure of the RRM domain of mouse Nup35 at 2.7 A resolution. The Nup35 RRM domain monomer adopts the characteristic betaalphabetabetaalphabeta topology, as in other reported RRM domains. The structure allowed us to locate the atypical RNP1 and RNP2 motifs. Among the RNP motif residues, those on the beta-sheet surface are different from those of the canonical RRM domains, while those buried in the hydrophobic core are highly conserved. The RRM domain forms a homodimer in the crystal, in accordance with analytical ultracentrifugation experiments. The beta-sheet surface of the RRM domain, with its atypical RNP motifs, contributes to homodimerization mainly by hydrophobic interactions: the side-chain of Met236 in the beta4 strand of one Nup35 molecule is sandwiched by the aromatic side-chains of Phe178 in the beta1 strand and Trp209 in the beta3 strand of the other Nup35 molecule in the dimer. This structure reveals a new homodimerization mode of the RRM domain.     

Permissive transmembrane helix heterodimerization is required for the expression of a functional integrin

The current paradigm is that integrin is activated via inside-out signalling when its cytoplasmic tails and TMs (transmembrane helices) are separated by specific cytosolic protein(s). Perturbations of the helical interface between the alpha- and beta-TMs of an integrin, as a result of mutations, affect its function. Previous studies have shown the requirement for specific pairing between integrin subunits by ectodomain-exchange analyses. It remains unknown whether permissive alpha/beta-TM pairing of an integrin is also required for pairing specificity and the expression of a functionally regulated receptor. We performed scanning replacement of integrin beta2-TM with a TM of other integrin beta-subunits. With the exception of beta4 substitution, others presented beta2-integrins with modified phenotypes, either in their expression or ligand-binding properties. Subsequently, we adopted alphaLbeta2 for follow-on experiments because its conformation and affinity-state transitions have been well defined as compared with other members of the beta2-integrins. Replacement of beta2- with beta3-TM generated a chimaeric alphaLbeta2 of an intermediate affinity that adhered to ICAM-1 (intercellular adhesion molecule 1) but not to ICAM-3 constitutively. Replacing alphaL-TM with alphaIIb-TM, forming a natural alphaIIb/beta3-TM pair, reversed the phenotype of the chimaera to that of wild-type alphaLbeta2. Interestingly, the replacement of alphaLbeta2- with beta3-TM showed neither an extended conformation nor the separation of its cytoplasmic tails, which are well-reported hallmarks of an activated alphaLbeta2, as determined by reporter mAb (monoclonal antibody) KIM127 reactivity and FRET (fluorescence resonance energy transfer) measurements respectively. Collectively, our results suggest that TM pairing specificity is required for the expression of a functionally regulated integrin.     

GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of Abeta42

Processing of the amyloid precursor protein (APP) by beta- and gamma-secretases leads to the generation of amyloid-beta (Abeta) peptides with varying lengths. Particularly Abeta42 contributes to cytotoxicity and amyloid accumulation in Alzheimer's disease (AD). However, the precise molecular mechanism of Abeta42 generation has remained unclear. Here, we show that an amino-acid motif GxxxG within the APP transmembrane sequence (TMS) has regulatory impact on the Abeta species produced. In a neuronal cell system, mutations of glycine residues G29 and G33 of the GxxxG motif gradually attenuate the TMS dimerization strength, specifically reduce the formation of Abeta42, leave the level of Abeta40 unaffected, but increase Abeta38 and shorter Abeta species. We show that glycine residues G29 and G33 are part of a dimerization site within the TMS, but do not impair oligomerization of the APP ectodomain. We conclude that gamma-secretase cleavages of APP are intimately linked to the dimerization strength of the substrate TMS. The results demonstrate that dimerization of APP TMS is a risk factor for AD due to facilitating Abeta42 production.     

HTTM, a horizontally transferred transmembrane domain

Sequence analysis of vitamin K-dependent gamma-carboxylases (VKGC) has revealed the presence of a novel domain, HTTM (for horizontally transferred transmembrane) in its N terminus. In contrast to most known domains, HTTM contains four transmembrane regions. Its occurrence in eukaryotes, bacteria and archaea is probably caused by horizontal gene transfer rather than by early evolution. The conservation of VKGC catalytic sites also indicates an enzymatic function for the other family members.     

Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes

The NOTCH1 receptor is cleaved within its extracellular domain by furin during its maturation, yielding two subunits that are held together noncovalently by a juxtamembrane heterodimerization (HD) domain. Normal NOTCH1 signaling is initiated by the binding of ligand to the extracellular subunit, which renders the transmembrane subunit susceptible to two successive cleavages within and C terminal to the heterodimerization domain, catalyzed by metalloproteases and gamma-secretase, respectively. Because mutations in the heterodimerization domain of NOTCH1 occur frequently in human T-cell acute lymphoblastic leukemia (T-ALL), we assessed the effect of 16 putative tumor-associated mutations on Notch1 signaling and HD domain stability. We show here that 15 of the 16 mutations activate canonical NOTCH1 signaling. Increases in signaling occur in a ligand-independent fashion, require gamma-secretase activity, and correlate with an increased susceptibility to cleavage by metalloproteases. The activating mutations cause soluble NOTCH1 heterodimers to dissociate more readily, either under native conditions (n = 3) or in the presence of urea (n = 11). One mutation, an insertion of 14 residues immediately N terminal to the metalloprotease cleavage site, increases metalloprotease sensitivity more than all others, despite a negligible effect on heterodimer stability by comparison, suggesting that the insertion may expose the S2 site by repositioning it relative to protective NOTCH1 ectodomain residues. Together, these studies show that leukemia-associated HD domain mutations render NOTCH1 sensitive to ligand-independent proteolytic activation through two distinct mechanisms.     

A modified, dual reporter TOXCAT system for monitoring homodimerization of transmembrane segments of proteins

The TOXCAT assay system developed by Russ and Engelman [TOXCAT: a measure of transmembrane helix association in a biological membrane, Proc. Natl. Acad. Sci. USA 96 (1999) 863-868] provides an in vivo means of selecting for and evaluating the strength of interaction between identical transmembrane alpha-helices. In the course of utilizing TOXCAT to study the architecture of a sodium channel hNa(V)1.5, an apparently strong dimerization of two of its putative transmembrane segments was revealed. Following random mutagenesis of these regions, several amino acids critical for the observed dimerizations were identified. In order to develop a more efficient means of isolating mutations which specifically disrupt dimerization of these transmembrane segments without affecting their membrane-targeting properties, we developed a modification to the original TOXCAT design in which the C-terminal maltose binding protein moiety is replaced by the beta-lactamase. We show that this assay system is capable of simultaneously monitoring the integrity of the chimeric protein, its membrane insertion activity, and the ability of the transmembrane segment under study to dimerize.     

Autoinhibitory and other autoregulatory elements within the dynein motor domain

The dyneins are a family of microtubule motor proteins. The motor domain, which represents the C-terminal 2/3 of the dynein heavy chain, exhibits homology to the AAA family of ATPases. It consists of a ring of six related but divergent AAA+ units, with two substantial sized protruding projections, the stem, or tail, which anchors the protein to diverse subcellular sites, and the stalk, which binds microtubules. This article reviews recent efforts to probe the mechanism by which the dyneins produce force, and work from the authors' lab regarding long-range conformational regulation of dynein enzymatic activity.     

Identification of a new presenilin-dependent zeta-cleavage site within the transmembrane domain of amyloid precursor protein

Gamma-secretase cleavage of beta-amyloid precursor protein (APP) is crucial in the pathogenesis of Alzheimer disease, because it is the decisive step in the formation of the C terminus of beta-amyloid protein (Abeta). To better understand the molecular events involved in gamma-secretase cleavage of APP, in this study we report the identification of a new intracellular long Abeta species containing residues 1-46 (Abeta46), which led to the identification of a novel zeta-cleavage site between the known gamma- and epsilon-cleavage sites within the transmembrane domain of APP. Our data clearly demonstrate that the new zeta-cleavage is a presenilin-dependent event. It is also noted that the new zeta-cleavage site at Abeta46 is the APP717 mutation site. Furthermore, we show that the new zeta-cleavage is inhibited by gamma-secretase inhibitors known as transition state analogs but less affected by inhibitors known as non-transition state gamma-secretase inhibitors. Thus, the identification of Abeta46 establishes a system to determine the specificity or the preference of the known gamma-secretase inhibitors by examining their effects on the formation or turnover of Abeta46.     

D2 dopamine receptor homodimerization is mediated by multiple sites of interaction,including an intermolecular interaction involving transmembrane domain 4

In this study, we examined the mechanisms of intermolecular interaction involved in D2 dopamine receptor dimer formation to develop an understanding of the quaternary structure of G protein-coupled receptors. The potential role of two mechanisms was investigated: disulfide bridges and hydrophobic interactions between transmembrane domains. D2 dopamine receptor oligomers were unaffected by treatment with a reducing agent; however, oligomers of the D1 dopamine receptor dissociated following a similar treatment. This observation suggested that other forces such as hydrophobic interactions were more robust in the D2 receptor than in the D1 receptor in maintaining oligomerization. To elucidate which transmembrane domains were involved in the intermolecular hydrophobic interactions, truncation mutants were generated by successive deletion of transmembrane domains from amino and/or carboxyl portions of the D2 dopamine receptor. Immunoblot analyses revealed that all the fragments were well expressed but only fragments containing transmembrane domain 4 were able to self-associate, suggesting that critical areas for receptor dimerization resided within this transmembrane domain. Disruption of the helical structure of transmembrane domain 4 in a truncated receptor capable of forming dimers interfered with its ability to self-associate; however, a similar disruption of the transmembrane domain 4 helix structure in the full-length receptor did not significantly affect dimerization. These results indicated that there are other sites of interaction involved in D2 receptor oligomer assembly in addition to transmembrane domain 4.     

Bridging of anions by hydrogen bonds in nest motifs and its significance for Schellman loops and other larger motifs within proteins

The nest is a protein motif of three consecutive amino acid residues with dihedral angles 1,2‐α_Rα_L (RL nests) or 1,2‐α_Lα_R (LR nests). Many nests form a depression in which an anion or δ‐negative acceptor atom is bound by hydrogen bonds from the main chain NH groups. We have determined the extent and nature of this bridging in a database of protein structures using a computer program written for the purpose. Acceptor anions are bound by a pair of bridging hydrogen bonds in 40% of RL nests and 20% of LR nests. Two thirds of the bridges are between the NH groups at Positions 1 and 3 of the motif (N1N3‐bridging)—which confers a concavity to the nest; one third are of the N2N3 type—which does not. In bridged LR nests N2N3‐bridging predominates (14% N1N3: 75% N2N3), whereas in bridged RL nests the reverse is true (69% N1N3: 25% N2N3). Most bridged nests occur within larger motifs: 45% in (hexapeptide) Schellman loops with an additional 4 → 0 hydrogen bond (N1N3), 11% in Schellman loops with an additional 5 → 1 hydrogen bond (N2N3), 12% in a composite structure including a type 1β‐bulge loop and an asx‐ or ST‐ motif (N1N3)—remarkably homologous to the N1N3‐bridged Schellman loop—and 3% in a composite structure including a type 2β‐bulge loop and an asx‐motif (N2N3). A third hydrogen bond is a previously unrecognized feature of Schellman loops as those lacking bridged nests have an additional 4 → 0 hydrogen bond. Proteins 2014; 82:3023–3031. © 2014 Wiley Periodicals, Inc.     

Determinants within an 18-amino-acid U1A autoregulatory domain that uncouple cooperative RNA binding,inhibition of polyadenylation,and homodimerization

The human U1 snRNP-specific U1A protein autoregulates its own production by binding to and inhibiting the polyadenylation of its own pre-mRNA. Previous work demonstrated that a short sequence of U1A protein is essential for autoregulation and contains three distinct activities, which are (i) cooperative binding of two U1A proteins to a 50-nucleotide region of U1A pre-mRNA called polyadenylation-inhibitory element RNA, (ii) formation of a novel homodimerization surface, and (iii) inhibition of polyadenylation by inhibition of poly(A) polymerase (PAP). In this study, we purified and analyzed 11 substitution mutant proteins, each having one or two residues in this region mutated. In 5 of the 11 mutant proteins, we found that particular amino acids associate with one activity but not another, indicating that they can be uncoupled. Surprisingly, in three mutant proteins, these activities were improved upon, suggesting that U1A autoregulation is selected for suboptimal inhibitory efficiency. The effects of these mutations on autoregulatory activity in vivo were also determined. Only U1A and U170K are known to regulate nuclear polyadenylation by PAP inhibition; thus, these results will aid in determining how widespread this type of regulation is. Our molecular dissection of the consequences of conformational changes within an RNP complex presents a powerful example to those studying more complicated pre-mRNA-regulatory systems.     

Structure and functional relevance of the Slit2 homodimerization domain

Slit proteins are secreted ligands that interact with the Roundabout (Robo) receptors to provide important guidance cues in neuronal and vascular development. Slit–Robo signalling is mediated by an interaction between the second Slit domain and the first Robo domain, as well as being dependent on heparan sulphate. In an effort to understand the role of the other Slit domains in signalling, we determined the crystal structure of the fourth Slit2 domain (D4) and examined the effects of various Slit2 constructs on chick retinal ganglion cell axons. Slit2 D4 forms a homodimer using the conserved residues on its concave face, and can also bind to heparan sulphate. We observed that Slit2 D4 frequently results in growth cones with collapsed lamellipodia and that this effect can be inhibited by exogenously added heparan sulphate. Our results show that Slit2 D4–heparan sulphate binding contributes to a Slit–Robo signalling mechanism more intricate than previously thought.     

Analysis of the transmembrane domain of influenza virus neuraminidase, a type II transmembrane glycoprotein, for apical sorting and raft association

Influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. Previously, it was shown that the transmembrane domain (TMD) of NA provides a determinant(s) for apical sorting and raft association (A. Kundu, R. T. Avalos, C. M. Sanderson, and D. P. Nayak, J. Virol. 70:6508-6515, 1996). In this report, we have analyzed the sequences in the NA TMD involved in apical transport and raft association by making chimeric TMDs from NA and human transferring receptor (TR) TMDs and by mutating the NA TMD sequences. Our results show that the COOH-terminal half of the NA TMD (amino acids [aa] 19 to 35) was significantly involved in raft association, as determined by Triton X-100 (TX-100) resistance. However, in addition, the highly conserved residues at the extreme NH(2) terminus of the NA TMD were also critical for TX-100 resistance. On the other hand, 19 residues (aa 9 to 27) at the NH(2) terminus of the NA TMD were sufficient for apical sorting. Amino acid residues 14 to 18 and 27 to 31 had the least effect on apical transport, whereas mutations in the amino acid residues 11 to 13, 23 to 26, and 32 to 35 resulted in altered polarity for the mutant proteins. These results indicated that multiple regions in the NA TMD were involved in apical transport. Furthermore, these results support the idea that the signals for apical sorting and raft association, although residing in the NA TMD, are not identical and vary independently and that the NA TMD also possesses an apical determinant(s) which can interact with apical sorting machineries outside the lipid raft.     

Mutations within the putative membrane-spanning domain of the simian immunodeficiency virus transmembrane glycoprotein define the minimal requirements for fusion, incorporation, and infectivity

The membrane-spanning domain (MSD) of a number of retroviral transmembrane (TM) glycoproteins, including those from the human and simian immunodeficiency viruses (HIV and SIV), have been predicted to contain a charged arginine residue. The wild-type SIV TM glycoprotein is 354 amino acids long. The entire putative cytoplasmic domain of SIV (amino acids 193 to 354) is dispensable for virus replication in vitro, and such truncation-containing viruses are capable of reaching wild-type titers after a short delay. We show here that further truncation of eight additional amino acids to TM185 results in a protein that lacks fusogenicity but is, nevertheless, efficiently incorporated into budding virions. By analyzing a series of nonsense mutations between amino acids 193 and 185 in Env expression vectors and in the SIVmac239 proviral clone, a region of the SIV TM that contains the minimum requirement for glycoprotein-mediated cell-to-cell fusion and that for virus replication was identified. Virus entry and infectivity were evident in truncations to a minimum of 189 amino acids, whereas cell-cell fusion was observed for a protein of only 187 amino acids. Glycoprotein was efficiently incorporated into budding virions in truncations up to 185 amino acids, indicating that such proteins are membrane anchored and are transported to the cell surface. However, truncation of the TM to 180 amino acids resulted in a protein that displays a transport defect and may be retained in the endoplasmic reticulum. Based on our analyses of these mutants, an alternative model for the MSD of SIV is proposed. Our model suggests that membrane-imbedded charged residues can be neutralized by side-chain interactions with lipid polar head groups. As a consequence, the membrane-spanning region can be reduced by more than a helical turn. This new model accounts for the ability of truncations within the predicted MSD to remain membrane anchored and maintain biological activity.     

The transmembrane domain and PXXP motifs of ApoE receptor 2 exclude it from carrying out clathrin-mediated endocytosis

The low density lipoprotein (LDL) receptor family comprises several proteins with similar structures including the LDL receptor and apoE receptor 2 (apoER2). The human brain expresses two major splice variants of apoER2 mRNA, one of which includes an additional exon that encodes 59 residues in the cytoplasmic domain. This exon is absent from the LDL receptor and contains three proline-rich (PXXP) motifs that may allow apoER2 to function as a signal transducer. To investigate the role of this insert, we took advantage of the well characterized low density lipoprotein receptor pathway. Chimeras comprising the ectodomain and transmembrane domain of the LDL receptor fused to the cytoplasmic domain of apoER2 lacking the PXXP-containing residues are able to mediate clathrin-dependent endocytosis of LDL as effectively as cells expressing the LDL receptor but not if the PXXP insert is present in the protein. Although expressed on the cell surface, the PXXP-containing chimeric receptor is excluded from clathrin vesicles as judged by its failure to co-localize with adaptor protein-2 possibly due to interaction with intracellular adaptors or scaffolding proteins. Chimeras with the transmembrane domain of apoER2, predicted to be longer than that of the LDL receptor by several residues, fail to mediate endocytosis of LDL or to co-localize with adaptor protein-2 regardless of the presence or absence of the PXXP insert. Thus features of apoER2 that distinguish it as a signaling receptor, rather than as an endocytosis receptor like the LDL receptor, reside in or near the transmembrane domain and in the proline-rich motifs.     

Signaling by chimeric erythropoietin-TGF-beta receptors: homodimerization of the cytoplasmic domain of the type I TGF-beta receptor and heterodimerization with the type II receptor are both required for intracellular signal transduction.

Transforming growth factor-beta (TGF-beta) affects multiple cellular functions through the type I and type II receptor Ser/Thr kinases (TbetaRI and TbetaRII). Analysis of TGF-beta signaling pathways has been hampered by the lack of cell lines in which both TbetaRI and TbetaRII are deleted, and by the inability to study signal transduction by TbetaRI independently of TbetaRII since TbetaRI does not bind TGF-beta directly. To overcome these problems, we constructed and expressed chimeric receptors with the extracellular domain of the erythropoietin receptor (EpoR) and the cytoplasmic domains of TbetaRI or TbetaRII. When expressed in Ba/F3 cells, which do not express EpoR, Epo induces the formation of a heteromeric complex between cell surface EpoR-TbetaRI and EpoR-TbetaRII chimeras. Neither the EpoR-TbetaRI nor the EpoR-TbetaRII chimera interacts with endogenous TGF-beta receptors. Ba/F3 cells expressing both EpoR-TbetaRI and EpoR-TbetaRII chimeras, but not EpoR-TbetaRI or EpoR-TbetaRII alone, undergo Epo-induced growth arrest. When expressed in Ba/F3 cells in the absence of the EpoR-TbetaRII chimera, EpoR-TbetaRI(T204D), a chimeric receptor with a point mutation in the GS domain of TbetaRI that is autophosphorylated constitutively, triggers growth inhibition in response to Epo. Thus, both homo- and heterodimerization of the cytoplasmic domain of the type I TGF-beta receptor are required for intracellular signal transduction leading to inhibition of cell proliferation. These chimeric receptors provide a unique system to study the function and signal transduction of individual TGF-beta receptor subunits independently of endogenous TGF-beta receptors.     

Role of the Vam3p transmembrane segment in homodimerization and SNARE complex formation

Intracellular membrane fusion in eukaryotic cells is mediated by SNARE (soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor) proteins and is known to involve assembly of cognate subunits to heterooligomeric complexes. For synaptic SNAREs, it has previously been shown that the transmembrane segments drive homotypic and support heterotypic interactions. Here, we demonstrate that a significant fraction of the yeast vacuolar SNARE Vam3p is a homodimer in detergent extracts of vacuolar membranes. This homodimer exists in parallel to the heterooligomeric SNARE complex. A Vam3p homodimer also formed from the isolated recombinant protein. Interestingly, homodimerization depended on the transmembrane segment. In contrast, formation of the quaternary SNARE complex from recombinant Vam3p, Nyv1p, Vti1p, and Vam7p subunits did not depend on the transmembrane segment of Vam3p nor on the transmembrane segments of its partner proteins. We conclude that Vam3p homodimerization, but not quaternary SNARE complex formation, is promoted by TMS-TMS interaction. As the transmembrane segments of Vam3p and other SNARE homologues were previously shown to be critical for membrane fusion downstream of membrane apposition, our results may shed light on the functional significance of SNARE TMS-TMS interactions.