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PNAS PLUS Lipid interaction of the C terminus and association of the transmembrane segments facilitate atlastin-mediated homotypic endoplasmic reticulum fusion Tina Y. Liua,b,1, Xin Bianc,d,1, Sha Sunc,d, Xiaoyu Huc,d, Robin W. Klemma,b, William A. Prinze, Tom A. Rapoporta,b, and Junjie Huc,d,2 a Howard Hughes Medical Institute and bDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115; cDepartment of Genetics and Cell Biology, College of Life Sciences, Nankai University, and dTianjin Key Laboratory of Protein Sciences, Tianjin 300071, China; and eLaboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892 The homotypic fusion of endoplasmic reticulum (ER) membranes is mediated by atlastin (ATL), which consists of an N-terminal cytosolic domain containing a GTPase module and a three-helix bundle followed by two transmembrane (TM) segments and a Cterminal tail (CT). Fusion depends on a GTP hydrolysis-induced conformational change in the cytosolic domain. Here, we show that the CT and TM segments also are required for efficient fusion and provide insight into their mechanistic roles. The essential feature of the CT is a conserved amphipathic helix. A synthetic peptide corresponding to the helix, but not to unrelated amphipathic helices, can act in trans to restore the fusion activity of tailless ATL. The CT promotes vesicle fusion by interacting directly with and perturbing the lipid bilayer without causing significant lysis. The TM segments do not serve as mere membrane anchors for the cytosolic domain but rather mediate the formation of ATL oligomers. Point mutations in either the C-terminal helix or the TMs impair ATL’s ability to generate and maintain ER morphology in vivo. Our results suggest that protein–lipid and protein–protein interactions within the membrane cooperate with the conformational change of the cytosolic domain to achieve homotypic ER membrane fusion. | | membrane perturbation content mixing organelle shaping organelle remodeling hereditary spastic paraplegia | T | he fusion of cellular membranes is critical for many biological processes. Homotypic fusion, i.e., the merging of identical membranes, is required for the remodeling of organelles, including the endoplasmic reticulum (ER) and mitochondria. Both organelles contain membrane tubules that are connected into a network by homotypic fusion (1, 2). Much less is known about this process than about heterotypic fusion, which occurs between viral and cellular membranes and between intracellular transport vesicles and target membranes. In viral fusion, the membranes are pulled together by an irreversible conformational change of a single protein (3, 4). In fusion during vesicular transport, three target (t-SNARE) proteins in one membrane and a vesicle (vSNARE) partner in the other zipper up to form a four-helix bundle in the fused lipid bilayer, a process facilitated by additional proteins (5–8). The homotypic fusion of ER membranes in metazoans is mediated by the atlastins (ATLs) (9, 10), a class of membranebound GTPases that belong to the dynamin family (11). The ATLs contain an N-terminal cytosolic domain consisting of a GTPase module and a three-helix bundle (3HB), as well as two closely spaced transmembrane (TM) segments and a C-terminal tail (CT) (Fig. 1A). A role for the ATLs in ER fusion is suggested by the observation that the depletion of ATLs leads to long, unbranched ER tubules in tissue culture cells (9) and to ER fragmentation in Drosophila melanogaster (10) possibly caused by insufficient fusion between the tubules. Nonbranched ER tubules also are observed upon expression of dominant-negative www.pnas.org/cgi/doi/10.1073/pnas.1208385109 ATL mutants (9, 12). In addition, antibodies to ATL inhibit ER network formation in Xenopus egg extracts (9). Finally, proteoliposomes containing purified Drosophila ATL undergo GTPdependent fusion in vitro (10, 13). ATL-mediated homotypic fusion of ER membranes appears to be physiologically important, because mutations in human ATL1, the major isoform in neuronal tissues, can cause hereditary spastic paraplegia (HSP) (14). HSP is a neurodegenerative disease caused by the shortening of the axons in corticospinal motor neurons, leading to progressive spasticity and weakness of the lower limbs. Yeast and plant cells do not possess ATLs, but similar GTPases—Sey1p in Saccharomyces cerevisiae and Root Hair Defective 3 (RHD3) in Arabidopsis thaliana—may have an analogous function (9). Mutations in the plant homolog RHD3 cause ER morphology defects (15) similar to those seen after depletion of ATLs. The tubular ER network in yeast is disrupted when the cells lack both Sey1p and one of the tubule-shaping proteins, Rtn1p or Yop1p (9). Normal ER morphology can be reestablished by expression of wild-type Sey1p (9) or human ATL1 (16), supporting the idea that these proteins are functional orthologs. The homotypic fusion of outer mitochondrial membranes also is mediated by membrane-bound GTPases of the dynamin family, the mitofusins in mammals and Fzo1p in yeast (17, 18). Thus, mitochondrial fusion may be achieved by a similar mechanism. Recent structural and biochemical studies provide significant insight into the mechanism of ATL-mediated homotypic fusion (13, 19). The N-terminal cytosolic domain of human ATL1 forms a nucleotide-dependent dimer in which the GTPase domains face each other (Fig. S1). In one crystal structure, the 3HBs following the GTPase domains point in opposite directions. This structure likely corresponds to a prefusion state in which the fulllength proteins still are anchored in different membranes. In another structure, the 3HBs are parallel to one another and have crossed over to dock against the GTPase domain of the partner molecule. In the full-length ATL, the two molecules would sit in the same membrane, likely representing a postfusion state. The conformational change between the pre- and postfusion states, which is triggered by Pi release during the GTP hydrolysis cycle Author contributions: T.Y.L., X.B., T.A.R., and J.H. designed research; T.Y.L., X.B., S.S., X.H., R.W.K., and W.A.P. performed research; T.Y.L., X.B., S.S., W.A.P., T.A.R., and J.H. analyzed data; and T.Y.L., T.A.R., and J.H. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. 1 T.Y.L. and X.B. contributed equally to this work. 2 To whom correspondence should be addressed. E-mail: [email protected]. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1208385109/-/DCSupplemental. PNAS Early Edition | 1 of 9 CELL BIOLOGY Edited by William T. Wickner, Dartmouth Medical School, Hanover, NH, and approved June 14, 2012 (received for review May 18, 2012) 3HB B TM2 N C GTPase TM1 CT N C Cytosol ER lumen % Total NBD Fluorescence A 20 15 WT 10 W490D L482D A486D L489D Tailless 5 0 0 10 20 30 Time [min] C 40 D 472 C 519 dmATL SGELSDFGGKLDDFATLLWEKFMR—PIYHGCMEKGIHHVATHATEMAVG hsATL1 mmATL1 xlATL2 drATL3 ceATLa cqATL SGEYRELGAVIDQVAAALWDQGSTNEALYKLYSAAATHRHLYHQAFPTP SGEYRELGAVIDQVAAALWDQGSTNEALYKLYSAAATHRHLCHQAFPAP SGEFRELGTLIDQIAEIIWEQLLK—PLSDNLMEDNIRQTVRNSIKAGLT SGHYREVGTAIDQATGVILEQ------ATDVLNKTRNQ----------SGHLREAGGYVDDAVTYVWTNFISPNANHLGPLGGAIQMGEALGAGNRT SGELSDFGVRLDEIANLLWENVSWFASCFCLTGLGIVLYVHQDELVYAL * N * * * * Fig. 1. An amphipathic helix in the CT of ATL facilitates fusion. (A) Domain structure and membrane topology of ATL. 3HB, three-helix bundle; TM1 and TM2, transmembrane segments; CT, C-terminal tail. (B) Full-length wild-type Drosophila ATL, ATL lacking the CT (tailless, residues 1–476), and ATL with point mutants in the CT region were reconstituted at equal concentrations into donor and acceptor vesicles. GTP-dependent fusion of donor and acceptor vesicles was monitored by the dequenching of an NBD-labeled lipid present in the donor vesicles. In all cases, fusion was initiated by addition of GTP. (C) Sequence alignment of the CTs from various ATLs. Predicted helices (orange and green cylinders) are indicated, as are the residue numbers for Drosophila ATL. Residues on the hydrophobic face of the first, amphipathic helix are enclosed in brown boxes. The sequence of the synthetic C-terminal peptide used in the following figures is underlined. ce, Caenorhabditis elegans; cq, Culex quinquefasciatus; dm, D. melanogaster; dr, Danio rerio; hs, Homo sapiens; mm, Mus musculus; xl, Xenopus laevis. (D) Helical wheel representation of the first helix (478–495) was generated using program HeliQuest (http://heliquest.ipmc.cnrs.fr/). Hydrophobic, negatively charged, and positively charged residues are shown in yellow, red, and blue, respectively. The termini are indicated, and relatively conserved residues on the hydrophobic face are indicated by asterisks. (13), would pull the membranes into close proximity so that they can fuse. The energy gained from the conformational change in the Nterminal cytosolic domain of ATL appears to be relatively small, because the interaction surface areas are not dramatically different in the pre- and postfusion states. This observation raises the possibility that domains missing in the crystal structures, i.e., the TM segments and the CT, also could play important roles in the fusion process. In fact, deletion of the CT reduces the fusion activity of ATL in vitro (13, 20). Here, we show that an amphipathic helix in the CT interacts with the lipid bilayer and destabilizes it to promote bilayer fusion with little membrane lysis. We also show that the TM segments mediate nucleotide-independent oligomerization of ATL molecules and play an essential role in fusion. Both the CT and TM segments are important for ATL’s ability to generate and maintain ER morphology in vivo. Results Amphipathic Helix in the CT Facilitates Fusion. To test the role of ATL’s CT in membrane fusion, we first used an in vitro lipidmixing assay. In this assay, purified wild-type or mutant Drosophila ATL is reconstituted into donor and acceptor proteoliposomes at a protein:lipid ratio of 1:2,000. The donor vesicles contain lipids labeled with 7-nitrobenzoxadiazole (NBD) and its FRET acceptor, rhodamine. NBD fluorescence is quenched initially by rhodamine in the proteoliposomes, but subsequent fusion of labeled with unlabeled acceptor proteoliposomes results in dilution of the fluorophores and dequenching of NBD. In agreement with previous results (10, 13), wild-type ATL gave efficient fusion in the presence of GTP and Mg2+, whereas a mutant lacking the entire CT was much less active (Fig. 1B). The CT is not absolutely essential, because the tailless ATL mutant promoted fusion at a fivefold higher concentration (Fig. S2A). To identify the region in the CT that is responsible for 2 of 9 | www.pnas.org/cgi/doi/10.1073/pnas.1208385109 enhancement of fusion activity, we compared the sequences of ATLs from different species. The only region of significant sequence conservation immediately follows the second TM segment and is predicted to form an amphipathic helix, which in Drosophila ATL comprises residues 478–502 (Fig. 1 C and D). Several residues on the hydrophobic face of the helix are similar across species (Fig. 1C). To dissect the function of the amphipathic helix, we tested the effect of point mutations on ATL-mediated membrane fusion. Mutations on the hydrophobic face of the helix, L482D, A486D, and L489D, reduced fusion to a level similar to that seen with the tailless mutant (Fig. 1B). The W490D mutation also reduced fusion, albeit less strongly. The hydrophilic face of the helix appears to be less important, because changing charged residues in the helix (residues D477, D483, E491) to Ala had little effect (Fig. S2C). Thus, the critical part of the CT for ATL-mediated fusion appears to be the hydrophobic residues at the N-terminal part of the helix. We made the striking observation that a synthetic peptide corresponding to the helix (CTH, residues 479–507 of Drosophila ATL) (Fig. 1C) could act in trans to rescue the fusion activity of the tailless mutant (Fig. 2A). Almost wild-type levels of fusion activity were reached with a peptide concentration of about 15 μM, corresponding to a molar ratio between the peptide and tailless ATL of ∼50:1. The requirement of such a high ratio is expected, because the two partners normally are covalently linked and thus are in close proximity. The CTH also stimulated lipid mixing by full-length wild-type ATL, indicating that CTH does not compete with the endogenous CT in the lipid-mixing reaction (Fig. 2A). Although the peptide alone caused only minor dequenching, significant lipid mixing was absolutely dependent on GTP hydrolysis and was not seen with GDP or GTPγS (Fig. 2A). Similar results were obtained with a slightly shorter peptide (residues 481–507), whereas a significantly shorter peptide (residues 479–494) required higher concentrations for transactivation Liu et al. PNAS PLUS B Tailless + CTH + GTP 15 WT + CT Tailless + CTH + GDP Tailless + CTH + GTP S Tailless + CTH Tailless + GTP 10 CTH 5 0 0 10 20 30 40 Time [min] 50 60 70 25 20 Tailless + CTH + GTP GTP 15 Tailless + D-CTH + GTP 10 CTH, L482D or A486D 0 0 C 10 20 30 40 Time [min] 50 D GTP 250 0 0 20 40 60 Time [min] 80 100 Tailless Dithionite CTH 500 120 Tailless 750 WT + CTH + GTP WT + GTP Tailless + CTH + GTP WT + CTH Tailless + CTH Tailless + GTP Tailless Detergent Tailless (0 min) Dithionite 0 GTP _ _ _ GDP _ _ + + _ 100 80 60 Tailless + CTH 1250 1000 Tailless + CTH 1500 Radius [nm] Relative Fluorescence Units Tailless + GTP Tailless + CTH (L482D) + GTP Tailless + CTH (A486D) + GTP 5 60 + _ + _ 70 40 20 _ _ _ + + _ + _ Fig. 2. Transactivation of tailless ATL fusion by a synthetic peptide. (A) The fusion of vesicles containing wild-type or tailless ATL (residues 1–476) was determined by lipid mixing in the absence or presence of a synthetic peptide corresponding to the amphipathic helix (CTH, residues 479–507 of Drosophila ATL; see Fig. 1C). When indicated, the peptide was added at 10 min and the nucleotide was added at 30 min. Controls without nucleotide are shown for comparison. (B) As in A, but with mutant peptides or a peptide consisting of D-amino acids (D-CTH). The slightly lower activity of D-CTH might indicate a small alteration in lipid interaction caused by the chiral nature of the phospholipids. (C) As in A, but with 1 mM sodium dithionite added twice before the reaction and with raw fluorescence data shown. (D) The effective hydrodynamic radii of vesicles containing tailless ATL at a protein:lipid ratio of 1:1,000 were determined by dynamic light scattering with and without the addition of the indicated peptides. The reactions were started at 37 °C by the addition of GTP and terminated after 10 min by the addition of EDTA, before size analysis. One representative experiment is shown, with the radii given as the average of three instrument readings. Error bars indicate the SE. (Fig. S2D). To test whether transactivation mimics the physiological fusion reaction, we introduced mutations in CTH that impaired fusion activity in full-length ATL. Indeed, peptides containing the L482D and A486D mutations did not stimulate fusion by tailless ATL (Fig. 2B). Because the lipid mixing we observed may be caused by fusion of only the outer leaflets of the bilayer, we added dithionite before the fusion reaction to test whether the inner leaflets also fuse. Dithionite is poorly membrane permeable and therefore reacts selectively with and abolishes the fluorescence of NBD in the outer leaflet of the bilayer. Thus, any dequenching of NBD after dithionite treatment results from inner leaflet mixing. Wildtype ATL induced inner leaflet mixing, in agreement with previous results (10), whereas tailless ATL alone was inactive but was activated by the addition of CTH in trans (Fig. 2C). As before, CTH also stimulated the fusion activity of wild-type ATL. To confirm CTH-stimulated fusion further, we used dynamic light scattering (DLS) to measure the radius of the proteoliposomes before and after fusion. Fusion was initiated by the addition of GTP and terminated by the addition of EDTA, which chelates the Mg2+ ions required for nucleotide binding and hydrolysis. EDTA also disassembles vesicles that are tethered to one another but not yet fused (10), so that an increase in proteoliposome size observed by DLS would reflect only fusion. As previously reported (10), vesicles containing wild-type ATL underwent a GTP-dependent size increase (Fig. S3). Although tailless ATL induced only a small increase in liposome size, a prominent size increase was observed when CTH was added in trans (Fig. 2D). Together, these results show that CTH stimulates the tailless ATL mutant to catalyze fusion of both the inner and outer leaflets of the lipid bilayer. Liu et al. We used peptide-stimulated fusion of tailless ATL to analyze the role of the amphipathic helix in greater detail. To test whether the helix functions through protein–protein interactions, we used a synthetic peptide consisting solely of D-amino acids; because of the reversed chirality, the peptide would not be expected to maintain its interactions with tailless ATL, if they existed. The D-amino acid CTH (D-CTH) greatly stimulated lipid mixing of the tailless mutant (Fig. 2B), suggesting that the helix likely does not act through protein–protein interactions. Liposome fusion monitored by DLS also showed that D-CTH has an activity similar to that of the L-amino acid CTH (Fig. 2D). In contrast, only small increases in the average liposome radius were caused by the addition of mutant peptides, L482D and A486D, as expected from the lipid-mixing experiments (Fig. 2B). We next tested whether the C-terminal helix of ATL inserts into the lipid bilayer, as has been observed with other amphipathic helices (21). To this end, we monitored the fluorescence of the single Trp residue in the CTH (Trp490 in ATL). Upon addition of liposomes, a slight blue shift was observed, indicating movement of the Trp residue into a more hydrophobic environment (Fig. S4). To test directly whether the Trp residue comes in contact with the hydrophobic tails of lipids, we added the peptide to liposomes containing lipids with doxyl groups in the hydrocarbon chain; the doxyl groups are expected to quench the Trp fluorescence upon contact. Indeed, quenching was observed with doxyl groups located at either position 5 or 12 in the hydrocarbon chain (Fig. 3A). The D-CTH also interacted with the lipid bilayer, whereas an L-amino acid peptide carrying the L482D or A486D mutation showed little binding to the membrane (Fig. 3A). PNAS Early Edition | 3 of 9 CELL BIOLOGY 20 30 Tailless + D-CTH WT + GTP Tailless + CTH (A486D) GTP, GDP or GTP S 25 Tailless + CTH 30 Tailless + CTH (L482D) WT + CTH + GTP % Total NBD Fluorescence % Total NBD Fluorescence A A F0 /F 1.15 1.05 1.00 CTH D-CTH CTH L482D CTH A486D L-Trp M.R.E. (deg cm2dmol-1) 1.10 0 -5000 CTH -10000 -15000 -20000 200 210 220 230 240 250 260 Wavelength (nm) 0 -5000 D-CTH -10000 -15000 -20000 200 210 220 230 240 250 260 Wavelength (nm) peptide Tailless + CTH Tailless Tailless + N-BAR Tailless + Sar1p Tailless + melittin 5 0 % Total Fluorescence % Total NBD Fluorescence D 20 10 5000 0 -5000 CTH L482D -10000 -15000 -20000 200 210 220 230 240 250 260 Wavelength (nm) 5000 C 15 M.R.E. (deg cm2dmol-1) 12-doxyl PC 5000 M.R.E. (deg cm2dmol-1) 5-doxyl PC 1.20 M.R.E. (deg cm2dmol-1) B 1.25 5000 0 -5000 CTH A486D -10000 -15000 -20000 200 210 220 230 240 250 260 Wavelength (nm) peptide + lipids 100 80 melittin CTH D-CTH reverse-CTH CTH (L482D) CTH (A486D) 60 40 20 0 0 10 20 Time [min] 30 40 0 1 2 3 Time [min] 4 5 Fig. 3. The amphipathic ATL helix interacts with the lipid bilayer. (A) C-terminal peptides [wild type (CTH) or mutants] or a peptide consisting of D-amino acids (D-CTH) were added to liposomes containing or lacking phosphatidylcholine with doxyl groups at position 5 or 12 of the hydrocarbon chain. The quenching of the fluorescence of Trp490 in the peptides was measured and is expressed as F0/F (maximal fluorescence with doxyl-free liposomes divided by maximal fluorescence with doxyl-containing liposomes). A control was done by adding the amino acid Trp instead of a peptide. Shown are the mean and SE of three experiments. (B) Circular dichroism spectra of wild-type, D-amino acid, and mutant CTH peptides were recorded in the absence (black lines) or presence (red lines) of liposomes. (C) The fusion of vesicles by tailless ATL was tested at a protein:lipid ratio of 1:1,000 in the presence of 10 μM of various amphipathic peptides: N-BAR helix (residues 1–24 of Rvs161p), Sar1p helix (residues 1–23 of Sar1p), and melittin. (D) The indicated peptides were added to liposomes loaded with calcein (peptide:lipid ratio 1:50), and the leakage of the dye was monitored by its dequenching. Circular dichroism measurements provided further evidence for an interaction of the amphipathic helix with lipids. The peptide corresponding to the wild-type sequence became more helical in the presence of liposomes (Fig. 3B). The D-peptide behaved similarly, although the sign of dichroism was reversed, as expected. In contrast, L-peptides containing the L482D or A486D mutations did not become more helical upon addition of liposomes (Fig. 3B). Taken together, these results show that the folding of the amphipathic helix is induced upon interaction with the lipid bilayer and that formation of the helix correlates with its ability to promote fusion. Surprisingly, other amphipathic peptides that are known to interact with lipid bilayers [including the curvature-inducing Nterminal helices of Sar1p and of the N-BAR domain protein amphiphysin (22–24)] did not promote the fusion activity of the tailless ATL in trans (Fig. 3C). The pore-forming melittin peptide (25) also showed only minimal transactivation, even when added at higher concentrations (Fig. S5). Specificity is supported further by the observation that a synthetic peptide corresponding to the reverse amino acid sequence of CTH (i.e., reading the sequence from the C to the N terminus), which is predicted to have an altered and shortened amphipathic helix (Fig. S6A), showed only low stimulation of fusion by tailless ATL (Fig. S6B). Using the doxyl-quenching assay, we also verified that each of these peptides indeed binds liposomes with composition similar to those used in the lipid-mixing assay (Fig. S7). These results suggest that CTH interacts with the lipid bilayer to stimulate ATL-mediated fusion in a different manner than these other amphipathic peptides. To test whether the amphipathic ATL helix destabilizes the lipid bilayer, we added the CTH to liposomes loaded with self4 of 9 | www.pnas.org/cgi/doi/10.1073/pnas.1208385109 quenching concentrations of the fluorescent dye calcein; perturbation of the bilayer would cause the release of calcein, resulting in dilution and a consequent increase in its fluorescence. Indeed, both the L- and D-amino acid CTH induced some calcein leakage from the liposomes at about the same peptide: lipid ratio as used in the fusion assay (Fig. 3D). In contrast, the peptides carrying the L482D or A486D mutations were inactive. The peptide with the reverse sequence also showed reduced activity (Fig. 3D), consistent with its behavior in the fusion assay (Fig. S6B). These results show that the activity of ATL’s C-terminal amphipathic helix is correlated with its ability to destabilize the integrity of the lipid bilayer. However, the helix is less potent than melittin in calcein leakage (Fig. 3D) but is much more active in stimulating the fusion of tailless ATL (Fig. 3C and Fig. S5), indicating it likely does not permeabilize membranes by forming pores. The fact that the CTH increases the permeability of lipid bilayers raises the possibility that it stimulates the fusion activity of tailless ATL by disrupting and reannealing the lipid bilayers rather than by promoting true fusion in which the permeability barrier between the inside and outside of the vesicles is maintained. To test this possibility, we used a content-mixing assay, in which two fluorophores, biotinylated R-phycoerythrin (RPE-biotin) and Cy5-labeled streptavidin (SA-Cy5), are encapsulated into donor and acceptor proteoliposomes, respectively (26). When vesicle contents mix during fusion or lysis, the interaction between the streptavidin and biotin moieties brings R-phycoerythrin and Cy5 close enough for the fluorophores to undergo FRET. Fusion can be distinguished from lysis by adding biotindextran (BDA) to the outside of the proteoliposomes, thus preventing FRET between leaked dyes. Maximum FRET fluoLiu et al. WT + GTP WT + GTP + BDA 40 30 GTP or GDP 20 10 WT WT + GDP WT + BDA WT + GDP + BDA 0 0 B 5 10 15 Time [min] 20 25 30 % Total RPE-biotin:SA-Cy5 FRET 60 Tailless + CTH + GTP 50 40 Tailless + GTP + CTH + BDA 30 GTP 20 CTH 10 Tailless + CTH Tailless + GTP Tailless + GTP + BDA Tailless Tailless + BDA Tailless + CTH + BDA 0 0 5 10 20 15 Time [min] 25 30 Fig. 4. CTH-stimulated fusion of tailless ATL involves mixing of vesicle contents. (A) Content mixing and leakage mediated by wild-type ATL was measured by FRET between RPE-biotin and SA-Cy5 encapsulated in donor and acceptor vesicles, respectively. GTP or GDP was added, as indicated. BDA with a molecular mass of 70 kDa was added where indicated to block the FRET contribution from content dyes released by membrane lysis. The data are presented as the increase in Cy5 fluorescence, expressed as a percentage of the total fluorescence determined after the addition of detergent in the absence of BDA. Nucleotide or buffer was added at 10 min. (B) As in A, but using vesicles reconstituted with tailless ATL. The CTH was added at 5 min, and GTP or buffer was added at 10 min. rescence is determined by adding detergent to a reaction performed in the absence of BDA. Lipid mixing is followed in parallel with content mixing by incorporating Marina Blue- and NBD-labeled lipids into donor vesicles and monitoring the dequenching of Marina Blue (26). We first tested whether wildtype ATL could induce content mixing during liposome fusion, because it has not yet been demonstrated that ATL mediates true, nonleaky fusion rather than just lipid mixing. Proper encapsulation of each dye into the proteoliposomes first was confirmed by adding one FRET partner outside proteoliposomes containing the other FRET partner; FRET occurred only when detergent was added to lyse the vesicles (Fig. S8 A and B). In the actual fusion reaction, wild-type ATL catalyzed GTP-dependent content mixing with nearly no lysis (Fig. 4A). The kinetics of content mixing corresponded to that of lipid mixing (Fig. S8C). Addition of the CTH to proteoliposomes containing tailless ATL induced significant nonleaky fusion as well, whereas almost no content mixing was observed in the absence of peptide or GTP (Fig. 4B). Again, GTP-dependent content mixing proceeded in parallel with lipid mixing (Fig. S8D). A small increase in FRET was observed in the content-mixing assay when CTH was added in the absence of GTP (Fig. 4B), indicating that the lipid binding of the peptide causes some leakage. Nevertheless, it is clear that the CTH does not induce massive lysis and primarily promotes nonleaky membrane fusion by tailless ATL, similar to the reaction with wild-type ATL. Liu et al. Analysis of the Role of TM Segments in Fusion. We next analyzed the role of the two closely spaced TM segments in promoting fusion, again using Drosophila ATL. To investigate whether the TM segments serve merely as membrane anchors, we replaced them with unrelated TMs and tested these mutants in the lipid-mixing assay. Replacement with the TM of the tail-anchored human Sec61β protein (ATL-Sec61β) resulted in a mutant with no detectable fusion activity (Fig. 6 A and B), although it was reconstituted into liposomes efficiently (Fig. S10D). A similar result was obtained when the TMs were replaced with the two TM segments of yeast Sac1p (ATL-Sac1p), which are separated by a loop of only ∼10 amino acids and thus form a hairpin structure similar to that in ATL. Mutant proteins lacking either TM1 and CT (cyt-TM2) or TM2 and CT (cyt-TM1) also were inactive (Fig. 6 A and C). Fusion of mutants lacking the CT (cyt-TM2, cytTM1, and ATL-Sec61β) was not restored when the CTH peptide was added in trans (Fig. S11A). Even when these mutants were reconstituted at a fivefold-higher ratio of protein:lipid, only cytTM2 showed some low fusion activity in the presence of the peptide (Fig. S11B). Taken together, these results indicate that the TM segments are not serving just as membrane anchors for the cytosolic domain during ATL-mediated membrane fusion. Furthermore, the distance between the two TM segments is important, because the insertion of seven residues (AAEEEEA) into the intervening loop (ATL-LL) rendered the protein inactive (Fig. 6C), even though membrane integration was not affected (Fig. S10D). To identify residues that are important for the function of the TM segments in membrane fusion, we mutated both conserved WT Tailless A474L L488A A511D Fig. 5. ATL function tested in vivo in yeast cells. Wild-type human ATL1 or the indicated mutants were expressed under the endogenous SEY1 promoter in S. cerevisiae cells lacking Sey1p and Yop1p (sey1Δ yop1Δ cells). See Fig. S9 for expression levels. The ER was visualized by expressing Sec63-GFP, focusing the microscope on either the periphery or the center of the cells. (Scale bars, 1 μm.) PNAS Early Edition | 5 of 9 PNAS PLUS CELL BIOLOGY 50 We next tested the role of the C-terminal amphipathic helix in vivo, taking advantage of the fact that human ATL1 can replace the functional ortholog Sey1p in S. cerevisiae; the abnormal cortical ER morphology in cells lacking Sey1p and the tubuleshaping protein Yop1p can be restored by expression of human ATL1 from a CEN plasmid under the constitutive MET25 promoter (16). Restoration of ER morphology also was observed when ATL1 was expressed at lower levels from the weaker endogenous SEY1 promoter (Fig. 5). The tailless ATL1 mutant or the A511D mutant (equivalent to A486D in Drosophila ATL) did not rescue the morphology defects (Fig. 5; for expression levels, see Fig. S9), indicating that the amphipathic helix is required for efficient ER network formation in vivo. As expected from the in vitro experiments (Fig. S2A), at higher expression levels (under the MET25 promoter), the tailless mutant was able to restore the tubular ER network (Fig. S2B). Periphery % Total RPE-biotin:SA-Cy5 FRET 60 Center A cyt-TM1 ATL-LL % Total NBD Fluorescence C B cyt-TM2 % Total NBD Fluorescence WT ATL-Sac1p D 24 19 WT ATL-LL cyt-TM1 cyt-TM2 14 9 4 -1 0 10 20 Time [min] 30 40 % Total NBD Fluorescence A 24 19 14 WT ATL-Sec61 ATL-Sac1p 9 4 -1 0 10 20 Time [min] 30 40 24 19 WT L445A A449L G444L L463A 14 9 4 -1 0 10 20 Time [min] 30 40 Fig. 6. Testing TM mutants of ATL in the fusion assay. (A) Schematic representation of the ATL constructs used. TM1 and TM2 are colored in yellow and orange, respectively, and the CT is colored in red. The membrane orientation of the TM segments is indicated by arrows. The TMs of Sac1p are shown in cyan and blue, and the TM of Sec61β is shown in green. (B) Fusion assays with wild-type ATL or TM replacement mutants. (C) As in B, but with TM deletion or insertion mutants. (D) As in B, but with point mutants in the TM segments. and less conserved amino acids (Fig. S12). Substitutions were made to nonpolar residues to avoid the possibility that the mutant proteins simply would be compromised in membrane integration. Although many mutants behaved like the wild-type protein or had moderately reduced fusion activity (e.g., L445A or A449L, respectively), the G444L and L463A mutants were almost inactive (Fig. 6D; see Fig. S12 for the effect of all mutations tested). Both the A449L and L463A mutants also were defective in vivo, because the corresponding human ATL1 mutants (A474L and L488A) did not restore normal ER morphology in yeast cells lacking Sey1p and Yop1p (Fig. 5). Taken together, these results indicate that the specific amino acid sequence of the TM segments is important for their function in membrane fusion. One potential function for the TMs is to mediate the oligomerization of ATL molecules, a possibility suggested by the observation that the overexpression of a fragment containing the TMs and CT of ATL causes a dominant-negative effect on ER morphology (9). Indeed, ATL molecules form oligomers in digitonin, but they dissociate in SDS or Triton X-100 (Fig. S13). This oligomerization is not mediated by the GTPase domains, because it occurs even when nucleotide binding is prevented by the addition of EDTA. To test directly whether the TM segments of ATL interact within the membrane, we performed coimmunoprecipitation experiments. As described previously (12, 27), when Myc- and Flag-tagged full-length human ATL1 (Myc-/Flag-hsATL1 FL) was cotransfected into COS-7 cells, Myc antibodies were able to precipitate Flag-hsATL1 (Fig. 7 A and B, lane 18), and, conversely, Flag antibodies could precipitate Myc-hsATL1 (Fig. 7B, lane 5). EDTA was included during the incubation to prevent nucleotide binding and thus coimmunoprecipitation caused by dimerization through the GTPase domains. In contrast, when Myc- and Flag-tagged proteins were transfected individually into COS-7 cells, and the extracts were mixed, no coimmunoprecipitation was observed (Fig. 7B, lanes 10 and 23). The interaction between ATL molecules was restored when GTPγS and MgCl2 were added to allow GTP-dependent dimerization of ATL molecules (Fig. 7B, lanes 15 and 28). These results indicate that 6 of 9 | www.pnas.org/cgi/doi/10.1073/pnas.1208385109 nucleotide-independent oligomerization requires that both partners reside in the same membrane. An Myc-tagged construct containing only the TMs and CT of human ATL1 (hsATL1 TMCT) (Fig. 7A) also interacted with Flag-hsATL1 FL when the proteins were expressed in the same cells but not in different cells (Fig. 7C, lanes 3 and 5 versus lanes 8 and 10). Taken together, these results suggest that the TMs mediate the nucleotide-independent association of ATL molecules. A recent report suggested that the 3HB, and not the TMs, are involved in oligomerization of Drosophila ATL (28). In agreement with this report, we found that Myc- and Flag-tagged constructs containing the 3HB, TMs, and CT associated with one another (Fig. 7D, lanes 28 and 30). However, in contrast to Pendin et al. (28), we found robust self-association with a construct containing only the TMs and CT (Fig. 7D, lanes 18 and 20). This self-association, together with evidence that the CT does not interact with other domains of ATL, suggests that the TMs indeed can mediate oligomerization. Furthermore, although Pendin et al. (28) found some self-association with a fragment containing the GTPase (G) domain and the 3HB, the interaction was very weak in their hands and was negligible in ours (Fig. 7D, lanes 23 and 25), suggesting only a minor contribution by the 3HB. Thus, our data indicate that the TMs are the major requirement for nucleotide-independent oligomerization of ATL molecules. Discussion Our results provide important insight into the mechanism of ATL-mediated homotypic ER fusion. We show that the previously identified GTPase-induced conformational change of the cytosolic domain is insufficient to achieve efficient fusion. Rather, both the CT and the TM segments play important and specific roles in membrane fusion. The crucial segment of the CT is a conserved amphipathic helix that follows immediately after the second TM segment. Although the amphipathic helix of the CT is not absolutely essential for fusion, it is physiologically important, because ATL mutants lacking the CT cause HSP (29–31), and tailless human ATL and a point mutant in the C-terminal helix are defective in Liu et al. PNAS PLUS A B C 3HB TM CT hsATL1 FL 1-558 TM-CT 449-558 G-3HB 3HB-TM-CT dmATL GTP S/Mg2+ 423-541 1-422 328-541 IB: Myc - - - - - - - - - - + + + + + 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Myc-hsATL1 FL Flag-hsATL1 FL IB: Flag 16 17 18 19 20 b IP oun a d un ntibo My IP un c an d tiFl ag un ad Myc-TM-CT & Flag-FL transfected individually lo ad un bo IP un a d un ntibo My IP un c an d tiFl ag Myc-TM-CT & Flag-FL co-transfected lo IB: Flag Flag-hsATL1 FL Myc-hsATL1 TM-CT IB: Myc lo 5 6 7 8 Myc-dmATL TM-CT IB: Myc 1 2 3 4 Flag-dmATL TM-CT IB: Flag 16 17 18 19 20 Myc-dmATL G-3HB 6 5 9 10 ad un bo IP un a d u n ntib o My IP un c an d tiFl ag 4 ad un bo IP un an d un ti-M bo y IP un c an d tiFl ag 3 ad un bo IP un a d un ntibo My IP un c an d tiFl ag D 2 lo 1 lo C * 21 22 23 24 25 26 27 28 29 30 7 8 9 10 Myc-dmATL 3HB-TM-CT 11 12 13 14 15 Flag-dmATL G-3HB * 21 22 23 24 25 CELL BIOLOGY G Myc- & Flag-FL transfected individually lo ad un bo IP un a d un ntib o My IP un c an d tiF lo ad lag un bo IP un a d un ntib o My IP un c a d lo ntia d Fl ag un bo IP un a d un ntib o My IP un c an d tiFl ag N Myc- & Flag-FL co-transfected Flag-dmATL 3HB-TM-CT 26 27 28 29 30 Fig. 7. The TMs of ATL mediate nucleotide-independent oligomerization. (A) Domain structure of ATL. The residue numbers of constructs used in coimmunoprecipitation are listed. dmATL, Drosophila melanogaster ATL; hsATL1, Homo sapiens ATL1. (B) Myc- and Flag-tagged hsATL1 were cotransfected into COS-7 cells and solubilized in digitonin or were transfected individually followed by mixing of the digitonin-solubilized cell extracts. Immunoprecipitation (IP) was performed with anti-Myc or anti-Flag antibodies. When indicated, 1 mM GTPγS and 5 mM MgCl2 were added. The samples were analyzed by SDS/PAGE and immunoblotting (IB) with anti-Myc or anti-Flag antibodies. Ten percent of the starting material (load) and of the material not bound to the antibodies (unbound) was analyzed also. (C) As in B, but with COS-7 cells expressing Myc-hsATL1 TM-CT and Flag-hsATL1. (D) As in B, but with COS-7 cells expressing Mycand Flag-tagged dmATL constructs. A contaminating Ig band from the antibody-conjugated matrix used in the IP is indicated by an asterisk. maintaining ER morphology in S. cerevisiae. Our results show that mutations in the helix dramatically reduce fusion activity and that the fusion defect of an ATL mutant lacking the C terminus is rescued when a synthetic peptide of the C-terminal amphipathic helix is added in trans. The peptide stimulates genuine GTP-dependent fusion of tailless ATL, as shown by lipid mixing in both leaflets of the bilayer, by a size increase of the proteoliposomes that was not caused by mere tethering of the vesicles, and, most importantly, by mixing of vesicle contents. The C-terminal helix likely does not function by interacting with other domains of the ATL molecule, because a CTH composed of D-amino acids also stimulates fusion in trans. In addition, the CTH stimulates the fusion of wild-type ATL, whereas an inhibitory effect might have been expected if it blocked the endogenous sequence from interacting with another domain of ATL. Using various biophysical assays, we show that instead the CTH interacts with lipids and becomes more helical upon contact with the lipid bilayer. The relevance of lipid interaction is supported by the observation that mutations in the CT of fulllength ATL that reduce fusion activity also affect lipid binding of the CTH. Given that both the N and C termini of the helix are on the cytosolic side of the membrane (12, 27), and that the most conserved part of the helix comprises only ∼15 amino acids, it seems likely that the helix sits in the cytosolic leaflet, with its hydrophobic residues inserted into the bilayer. The C-terminal helix probably does not promote fusion by forming a lipidic pore like melittin or by generating membrane curvature like the amphipathic helices in Sar1p and N-BAR, Liu et al. because these peptides do not mimic the activity of the CTH in the lipid-mixing assay. The ATL helix may function by destabilizing the lipid bilayer, because the CTH releases calcein from the vesicle interior but fusion-defective mutants do not. Although the exact mechanism by which the ATL helix disturbs the integrity of the lipid bilayer is unclear, we speculate that it displaces negatively charged phospholipid head groups and exposes the hydrocarbon chains of lipid molecules at the membrane surface, a perturbation that would lower the energy barrier for fusion between the approaching bilayers. The fact that the C-terminal helix destabilizes the lipid bilayer raised the question of whether ATL mediates genuine fusion or merely rupture and reannealing of the lipid bilayer. Here, we show that, indeed, wild-type ATL causes GTP-dependent mixing of vesicle contents without significant lysis. Even the combination of tailless ATL mutant and CTH mediates true fusion, although some leakiness was observed, consistent with the calcein release experiments. The increased lysis is likely caused by the high concentration of the CTH in this reaction; in wild-type ATL, the helix would be linked covalently to the rest of the protein and would act only locally at the site of fusion. The content-mixing data provide strong evidence that ATL is capable of mediating complete membrane fusion without significantly compromising the permeability barrier of the lipid bilayer. Maintaining the barrier during fusion would be important to retain luminal ER proteins and maintain stores of calcium ions in the ER lumen. PNAS Early Edition | 7 of 9 Our results indicate that the TM segments are even more important than the CT for ATL’s function. The TMs are more than just membrane anchors, because they cannot be replaced by unrelated TMs. In addition, even fairly conservative point mutations compromise fusion, highlighting their sequence-specific function. Several of these mutants also are defective in maintaining ER morphology in S. cerevisiae. One role for the TMs is to mediate nucleotide-independent oligomerization of ATL molecules, as shown by coimmunoprecipitation and sucrose gradient centrifugation experiments. We propose that the association between the TM segments clusters ATL molecules in the membrane before fusion. Our results suggest a refined model for ATL-mediated membrane fusion in which the CT and TMs of ATL cooperate with the N-terminal cytosolic domain. First, several ATL molecules in a membrane would associate with each other through their TM segments. Second, these complexes would interact with similarly assembled ATL molecules in another membrane; the interaction of ATL molecules across the two membranes would require GTP binding. It also is conceivable that the first and second steps are coordinated rather than occurring in a strictly consecutive manner. Third, GTP hydrolysis and phosphate release would trigger a conformational change that would pull the membranes toward each other for fusion. Because the energetic gain from the conformational change of a single pair of opposing ATL molecules may be relatively small, we speculate that nucleotideindependent oligomerization of ATL molecules would increase the efficiency of fusion by allowing several ATL molecules in each membrane to undergo synchronously the conformational changes leading to fusion. Local perturbation of the membrane bilayer by the CT also could contribute to the process by lowering the energy barrier for the approach and eventual merging of the membranes. Finally, once fusion has been completed and the postfusion conformation is reached, GDP would be released, allowing the nucleotide-dependent ATL dimers to dissociate and to start a new round of fusion. It has been suggested that fusion involves the oligomerization of ATL molecules in different membranes through their 3HBs (20, 28). Although it is possible that an interaction between the 3HBs of opposing ATL molecules occurs during the nucleotidedependent conformational change, our results show that the 3HB plays a minor or negligible role in the nucleotide-independent oligomerization of ATL molecules. Rather, the TMs mediate this association of ATL molecules in the same membrane before nucleotide binding. Our results on the mechanism of ATL-mediated fusion may be relevant to other fusion reactions as well. As in the ATLs, the assembly of the soluble domains of t- and v-SNAREs results in a relatively modest energy gain (32), indicating that other mechanisms, such as those described here for ATL, also may be necessary for efficient fusion. In fact, the v-SNARE protein synaptobrevin possesses a short helix adjacent to the TM segment that is believed to interact with the lipid bilayer before fusion (33, 34), similar to the C-terminal amphipathic helix of ATL. In flavivirus fusion proteins, a conserved amphipathic helix immediately preceding the TM segments also is postulated to interact with lipids at early phases of the fusion process (35, 36). The ability to use a synthetic peptide to activate fusion in trans allowed us to provide the best evidence yet that amphipathic helices affect membrane fusion by inserting into the lipid phase. In other fusion reactions, the TM segments also may be more than simply membrane anchors. The TM of the SNARE protein synaptobrevin appears to sit in the membrane at an angle and is believed to destabilize the lipid bilayer (34, 37), a result that is supported by experiments with a synthetic peptide (38). In addition, point mutations in the TMs of both syntaxin and synaptobrevin compromise fusion (34, 39), although perhaps not as strongly as mutations in ATL. Oligomerization of SNARE pro8 of 9 | www.pnas.org/cgi/doi/10.1073/pnas.1208385109 teins through their TM regions also has been reported, similar to our findings for ATL (for review, see ref. 40). In the case of viral fusion proteins, specific TM requirements vary (40). Replacing the TM of the HIV-1 gp41 protein with some unrelated TMs caused a reduction in fusion efficiency (41), whereas the replacement with another TM had no effect (42). In the case of vesicular stomatitis virus G protein (43) or influenza virus hemagglutinin (44), other TMs worked just as well as the native TM. On the other hand, point mutations in the TMs of these fusion proteins can reduce their activity (45, 46). Thus, it seems that often the TMs in other fusion reactions also may play a specific role, although perhaps they are not as crucial as in ATL. Finally, we propose that the mechanism of ATL-mediated fusion is shared by related GTPases involved in homotypic fusion. The functional orthologs of ATL in yeast and plants (Sey1p in S. cerevisiae and RHD3 in A. thaliana) have similar GTPase domains, a helical domain, and two closely spaced TM segments (9). They also contain a conserved amphipathic helix immediately following the second TM segment. The mitofusin/ Fzo1p involved in the fusion of the outer mitochondrial membrane also has a related GTPase domain, a coiled-coil domain, and two closely spaced TM segments. Although the overall mechanism therefore may be the same for all these GTPases, mitofusin/Fzo1p contains C-terminal heptad repeats instead of an amphipathic helix, and these repeats have been proposed to form antiparallel interactions with a mitofusin/Fzo1p molecule in the apposed membrane (47). This difference indicates that there may be variations in the common theme of homotypic membrane fusion. Materials and Methods Lipid-Mixing Assay. Full-length, codon-optimized Drosophila ATL was expressed in Escherichia coli as a GST fusion and was purified on glutathione agarose. The GST moiety was cleaved off by thrombin and removed with glutathione agarose. Detergent-mediated reconstitution was used to integrate ATL into preformed donor liposomes [82:15:1.5:1.5 mole percent of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS):NBD-1,2-dipalmitoyl-sn-glycero-3phosphoethanolamine (DPPE):rhodamine-DPPE] and acceptor liposomes (83.5:15:1.5 mole percent of POPC:DOPS:dansyl-DPPE) as described (10). The fusion assays were performed as described previously (13). For dithionite quenching, 1 μL 100 mM dithionite in buffer [25 mM Hepes (pH 10), 100 mM KCl, 10% (vol/vol) glycerol] was added during the lipid-mixing assay when indicated. The initial background fluorescence was subtracted from the raw fluorescence readings, and these values are expressed as percentages of the maximum fluorescence after detergent addition. Content-Mixing Assay. Fusion reactions were carried out as for lipid-mixing experiments, except that RPE-biotin was incorporated into the lumen of donor vesicles, and SA-Cy5 was incorporated into the lumen of acceptor vesicles (26). Content mixing and leakage were determined by FRET between RPE-biotin and SA-Cy5, and content mixing only was determined after the addition of BDA with a molecular mass of 70,000 Da to the outside of the vesicles, as previously described (26). Lipid mixing was determined in parallel by dequenching of the fluorescence of Marina Blue-PE (26). Dansyl-DPPE (excitation at 336 nm and emission at 517 nm) was included in the lipid mix of the acceptor vesicles to determine total lipid concentration, but it did not interfere with the fusion assays. All measurements were made using a SpectraMax M5 Microplate Reader (Molecular Devices, LLC). Thesit (C12E9) was added to reactions lacking BDA to determine the maximum fluorescence. The initial background fluorescence was subtracted from the raw fluorescence readings, and these values are expressed as percentages of the maximum fluorescence after detergent addition. Dynamic Light Scattering. Fusion reactions using proteoliposomes containing tailless or wild-type ATL were carried out using the procedures and conditions used in the lipid-mixing experiments. Peptide (15 μM) was added 10 min before GTP addition, where indicated. EDTA-containing buffer was added to stop vesicle fusion before size analysis. The mean effective hydrodynamic radii of proteoliposomes were determined using a DynaPro Nanostar instrument (Wyatt). Liu et al. Circular Dichroism. Circular dichroism experiments were performed on a Jasco J-815 instrument at 25 °C. Where indicated, 30 μM peptide in 10 mM potassium phosphate (pH 7.5), 100 mM KCl, and liposomes (85:15 mole percent POPC: DOPS, final concentration of 1 mM lipids) were included. Spectra were collected from 200–260 nm at a bandwidth of 1 nm and a scan speed of 100 nm/min. Mammalian Tissue Culture, Transfection and Coimmunoprecipitation. COS7 Cells were maintained at 37 °C with 5% CO2 in DMEM containing 10% (vol/ vol) FBS and were transfected using FuGENE HD (Roche). Coimmunoprecipitation was performed as described previously (9). Further details on methods are provided in SI Materials and Methods. Calcein-Leakage Assay. Liposomes (84.5:15:0.5 mole percent POPC:DOPS:Texas Red-DPPE) were prepared as described previously (10), except that calcein was encapsulated in the liposomes at a concentration of 100 mM. Then 2 μM peptide was added to 0.1 mM lipids in a 96-well plate, and the fluorescence (excitation at 490 nm, emission at 520 nm) was monitored at room temperature using a SpectraMax M5 Microplate Reader (Molecular Devices). ACKNOWLEDGMENTS. We thank A. Stein for helpful suggestions and R. King and A. Stein for critical reading of the manuscript. T.Y.L. is supported by a fellowship from the National Science Foundation, and R.W.K is supported by a European Molecular Biology Organization Long-Term Fellowship. W.A.P. is supported by the National Institute of Diabetes and Digestive and Kidney Diseases intramural program. T.A.R. is a Howard Hughes Medical Institute investigator. J.H. is supported by National Basic Research Program of China 973 Program, Grant 2010CB83370; National Science Foundation of China Grant 3097144; and an International Early Career Scientist grant from the Howard Hughes Medical Institute. 1. Baumann O, Walz B (2001) Endoplasmic reticulum of animal cells and its organization into structural and functional domains. Int Rev Cytol 205:149–214. 2. Hoppins S, Lackner L, Nunnari J (2007) The machines that divide and fuse mitochondria. Annu Rev Biochem 76:751–780. 3. Harrison SC (2008) Viral membrane fusion. Nat Struct Mol Biol 15:690–698. 4. 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Yeast cells lacking SEY1 and YOP1 were cultured and visualized as previously described (9). PNAS Early Edition | 9 of 9 CELL BIOLOGY Doxyl-Quenching Assay. Peptide was mixed with liposomes (84.5:15:0.5 mole percent POPC:DOPS:NBD-DPPE) at a peptide:lipid molar ratio of 1:40, and the fluorescence of Trp was measured. Emission intensity at the peak maximum in the presence of liposomes with or without doxyl-PC was used to determine the extent of quenching. Experiments were performed at room temperature on the SpectraMax M5 Microplate Reader (Molecular Devices). Supporting Information Liu et al. 10.1073/pnas.1208385109 SI Materials and Methods Constructs, Peptides, and Antibodies. Codon-optimized full-length Drosophila melanogaster atlastin (ATL) (residues 1–541) or the tailless mutant (residues 1–476) was cloned into pGEX-4T-3 or pGEX-6P-1. For the expression of human ATL1 in yeast at endogenous levels, the full coding region of ATL1 plus an N-terminal HA tag and 300-bp upstream sequences of SEY1 gene were amplified and cloned into YCplac111 (a LEU2/CEN plasmid). The insertion of the AAEEEEA sequence and the swapping of the Drosophila transmembrane (TM) region with that of human Sec61β (residues 61–97) or Saccharomyces cerevisiae Sac1p (residues 523–580) were achieved using PCR with overlapping primers. Point mutations were generated using the QuikChange Site-Directed Mutagenesis Kit (Stratagene). All constructs were confirmed by DNA sequencing. Peptides were synthesized by GL Biochem except for melittin, which was purchased from GenScript. The Xenopus ATL antibodies were generated in rabbits using the full-length protein as antigen. Protein Expression and Purification. Drosophila melanogaster ATL was expressed in Escherichia coli as a GST fusion, as described (1). The cells were lysed in A100 buffer [25 mM Hepes (pH 7.5), 100 mM KCl, 1 mM EDTA, 2 mM β-mercaptoethanol, and 10% (vol/vol) glycerol]. The membranes were pelleted by centrifugation and solubilized in Triton X-100. The GST fusion proteins were isolated with glutathione Sepharose beads (GE Healthcare), washed, and eluted with 10 mM glutathione in A100 buffer with 0.1% Triton X-100. The GST tag was cleaved by thrombin or Prescission protease (GE Healthcare) and was removed with glutathione agarose. Lipid-Mixing Assay. All in vitro lipid-mixing assays were performed as previously described (1). In brief, lipids [82:15:1.5:1.5 mole percent 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS): 7-nitrobenzoxadiazole (NBD)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE):rhodamine-DPPE for donor vesicles or 83.5:15:1.5 mole percent POPC:DOPS:dansyl-DPPE for acceptor vesicles] were dried down to a film, hydrated with A100 buffer, and extruded through polycarbonate filters with a pore size of 100 nm as described previously (2). Proteins were reconstituted at a protein:lipid molar ratio of 1:2,000, except in Fig. 3C, where the ratio was 1:1,000. The final lipid concentration in the lipid mixing reaction was 0.6 mM, with donor and acceptor liposomes added at a 1:3 ratio. Peptides were used at a concentration of 15 μM in the lipid-mixing assay, unless otherwise indicated. The fluorescence intensity of NBD was monitored with an excitation of 460 nm and emission of 538 nm. The initial NBD fluorescence was set to zero, and the maximum fluorescence was determined after addition of dodecyl maltoside. Dithionite-Quenching Assay. The dithionite quenching assays were performed using the same conditions as the lipid-mixing assays, except that 1 μL 100 mM dithionite in 25 mM Hepes (pH 10), 100 mM KCl, 10% (vol/vol) glycerol was added twice to 100 μL fusion reactions before peptide addition to selectively abolish NBD fluorescence on the outer leaflet of the bilayer. Content-Mixing Assay. Content-mixing was assayed using an approach developed previously for SNARE-mediated membrane fusion (3). Donor liposomes (82:15:1.5:1.5 mole percent POPC: DOPS:Marina Blue-PE:NBD-DPPE) and acceptor liposomes Liu et al. www.pnas.org/cgi/content/short/1208385109 (83.5:15:1.5 mole percent POPC:DOPS:dansyl-DPPE) were formed as for the lipid-mixing experiments. Reconstitution of ATL into proteoliposomes was done as described (2), except that 8 μM biotinylated R-phycoerythrin (RPE-biotin) (Invitrogen) was included during the reconstitution for donor vesicles and 8 μM Cy5-labeled streptavidin (SA-Cy5) (Invitrogen) was included for acceptor liposomes. Detergent removal by gradual addition of Bio-Beads SM-2 led to encapsulation of the dyes in the proteoliposomes. Unencapsulated content dyes were removed from the solution by incubation with NeutrAvidin resin (Pierce) for donor vesicles and biotin agarose (Sigma) for acceptor vesicles. The reaction was carried out as in the lipid-mixing assay, except that 20-μL volumes were used in 384-well plates. Content mixing and leakage was monitored by exciting RPE-biotin at 565 nm and detecting Cy5 fluorescence at 675 nm, and lipid mixing was monitored using dequenching of Marina Blue (excitation: 365 nm; emission: 460 nm). Biotin-dextran (1 μM) was included where indicated to block FRET resulting from contents that had been leaked from the vesicles during fusion. Maximum FRET caused by content mixing and leakage was determined by adding 1 μL of 10% (vol/vol) Thesit (C12E9; Affymetrix, Inc.) to reactions lacking biotin-dextran. Dynamic Light Scattering. Fusion reactions using proteoliposomes containing tailless or wild-type ATL were carried out using the procedures and conditions used in the lipid-mixing experiments. Peptide (15 μM) was added 10 min before GTP addition in all reactions. Reactions, which included 5 mM MgCl2, were diluted 1:2 into EDTA buffer (30 mM EDTA, 25 mM Hepes, 100 mM KCl, 10% (vol/vol) glycerol, 2 mM β-mercaptoethanol) to stop vesicle fusion. Effective hydrodynamic radii of proteoliposomes were determined using a DynaPro Nanostar instrument (Wyatt), which detects light scattered at 90° to the incident beam. Laser power and attenuation levels were set automatically by the instrument. The average of three measurements per sample was used to represent the average hydrodynamic radius of the vesicles. Doxyl-Quenching Assay. Liposomes (84.5:15:0.5 mole percent POPC:DOPS:NBD-DPPE) either with or without 10 molar per cent doxyl PC (Avanti Polar Lipids) were formed as described previously (2). Peptide was mixed with liposomes at a peptide:lipid molar ratio of 1:40. Trp fluorescence was excited at 280 nm, and emission spectra were taken from 310–410 nm. Blank spectra containing only liposomes were subtracted from the data. Emission intensity at the peak maximum in the presence of liposomes with or without doxyl-PC was used to determine the extent of quenching. Experiments were performed at room temperature on a SpectraMax M5 Microplate Reader (Molecular Devices). Circular Dichroism. Circular dichroism experiments were performed on a Jasco J-815 instrument. Each peptide was dissolved in 10 mM potassium phosphate (pH 7.5), 100 mM KCl and was analyzed in 1-mm path-length quartz cells at a concentration of 30 μM. Liposomes (85:15 mole percent POPC:DOPS), prepared as previously described (2), were included where indicated at a concentration of 1 mM. Spectra were collected at 25 °C from 200–260 nm. Each spectrum was the average of nine scans, performed at a bandwidth of 1 nm and a scan speed of 100 nm/min. Control spectra with buffer or liposomes only were subtracted from the corresponding peptide data. Calcein-Leakage Assay. Liposomes (84.5:15:0.5 mole percent POPC:DOPS:Texas Red-DHPE) were prepared as described 1 of 8 without glycerol), 300 μL of 100 mM carbonate buffer (pH 11.0), or 300 μL 0.1% Triton X-100 in B100 buffer and were centrifuged in a Beckman TLA 100.3 rotor at 200,000 × g for 75 min. The supernatants and pellets were analyzed by SDS/PAGE and immunoblotting with anti-Xenopus ATL antibodies. previously (2), except that the dried lipid film was hydrated with 100 mM calcein in 25 mM Hepes (pH 7.5), 10% (vol/vol) glycerol, 1 mM EDTA, and the vesicles then were extruded through filters with a pore size of 200 nm. Unincorporated calcein was separated from the liposomes using a Sephadex G50 column equilibrated in 25 mM Hepes (pH 7.5), 150 mM KCl, 10% (vol/vol) glycerol, 1 mM EDTA. Peptide was added to 0.1 mM lipids in a 96-well plate, and the fluorescence (excitation at 490 nm, emission at 520 nm) was monitored at room temperature using a SpectraMax M5 Microplate Reader (Molecular Devices). Control samples without peptide added were subtracted from the data. Sucrose Gradient Centrifugation. Full-length or mutant Drosophila ATL was reconstituted at a protein:lipid molar ratio of 1:2,000. Proteoliposomes (30 μL) were treated with 1% of the indicated detergents for 1 h at 4 °C and then were loaded on top of a 250 μL 5–25% (wt/vol) sucrose gradient prepared in B100 buffer. Where indicated, 5 mM EDTA was added also. The samples were centrifuged in a Beckman TLS-55 rotor at 174,000 × g for 2 h, fractionated, and analyzed by SDS/PAGE and immunoblotting with antibodies to Xenopus ATL. Fluorescence Microscopy in Yeast. Yeast cells lacking SEY1 and YOP1 were cultured and visualized as previously described (4). In brief, cells were imaged in growth medium with an Olympus BX61 microscope, UPlanApo 1003/1.35 lens, QImaging Retiga EX camera, and IVision version 4.0.5 software. Membrane-Association Assay. Proteoliposomes (10 μL) used for fusion were mixed with 300 μL of B100 buffer (A100 buffer Mammalian Culture, Transfection, and Coimmunoprecipitation. COS7 cells were maintained at 37°C with 5% CO2 in DMEM containing 10% (vol/vol) FBS and were transfected using FuGENE HD (Roche). Coimmunoprecipitation was performed as described previously (4). 1. Bian X, et al. (2011) Structures of the atlastin GTPase provide insight into homotypic fusion of endoplasmic reticulum membranes. Proc Natl Acad Sci USA 108: 3976–3981. 2. Orso G, et al. (2009) Homotypic fusion of ER membranes requires the dynamin-like GTPase atlastin. Nature 460:978–983. 3. Zucchi PC, Zick M (2011) Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis. Mol Biol Cell 22:4635–4646. 4. Hu J, et al. (2009) A class of dynamin-like GTPases involved in the generation of the tubular ER network. Cell 138:549–561. Pre-fusion Post-fusion GTPase-1 GTPase-1 3HB-1 GTPase-2 GTPase-2 3HB-2 3HB-2 3HB-1 Fig. S1. Structures of human ATL1. Structures of the dimers of the N-terminal cytosolic domain of human ATL1, corresponding to the pre- and postfusion states (Protein Data Bank ID codes 3QOF and 3QNU, respectively), are shown in cartoon representation. The GTPase domains are colored in yellow and cyan, and the three-helix bundles are shown in orange and blue. GDP is shown in green stick representation, and an Mg2+ ion is shown as a lime-colored sphere. The positions of the TM segments following the N-terminal cytosolic domain and of the membranes are shown for reference. Liu et al. www.pnas.org/cgi/content/short/1208385109 2 of 8 B 14 Tailless 1:400 Tailless 1:2000 9 4 -1 C 0 10 20 Time [min] 30 40 D 30 25 % Total NBD Fluorescence % Total NBD Fluorescence + Met25-tailless 19 Center % Total NBD Fluorescence 24 Periphery A 20 15 WT D477A D483A D491A 10 5 0 0 10 20 Time [min] 30 20 15 10 40 Tailless Tailless + short-CTH 5 0 0 10 20 Time [min] 30 40 WT (0 min) WT WT WT Fig. S2. Membrane fusion with C-terminal tail (CT) mutants of ATL. (A) The purified tailless ATL mutant (residues 1–476) was reconstituted into liposomes at a protein:lipid molar ratio of 1:400 or 1:2,000 and was tested in the fusion assay. Note that the tailless ATL mutant promotes membrane fusion at higher concentrations. (B) The endoplasmic reticulum (ER) morphology was analyzed in S. cerevisiae cells lacking Sey1p and Yop1p (sey1Δ yop1Δ cells) in the presence or absence of tailless human ATL1. The tailless mutant was expressed under the MET25 promoter. The ER was visualized by expressing Sec63-GFP, focusing the microscope on either the periphery or the center of the cells. (Scale bar, 1 μm.) (C) ATL with point mutants in the CT region was reconstituted at a protein:lipid molar ratio of 1:1,000 and was tested in the fusion assay. (D) The fusion of vesicles containing tailless ATL was determined in the absence or presence of a synthetic peptide corresponding to the N-terminal part of the amphipathic helix (short-CTH; residues 479–494 of Drosophila ATL) at 45 μM. 0 GTP _ _ _ GDP _ _ 120 Radius [nm] 100 80 60 40 20 + + _ Fig. S3. Fusion with wild-type ATL measured by dynamic light scattering. Vesicles containing wild-type ATL at a protein:lipid ratio of 1:400 were incubated with GTP or GDP or without nucleotide for 10 min. The reactions were terminated after 10 min by the addition of EDTA, followed by analysis by dynamic light scattering. One representative experiment is shown, with the radii given as the average of three instrument readings. Error bars indicate SE. Liu et al. www.pnas.org/cgi/content/short/1208385109 3 of 8 Fluorescence (arbitrary units) 6000 CTH + lipids 5000 CTH 4000 3000 2000 1000 0 310 320 330 340 350 360 370 Wavelength (nm) 380 390 400 Fig. S4. Fluorescence spectrum of the CTH peptide. The fluorescence of the single Trp490 residue in the CTH peptide was monitored in the presence and absence of liposomes (84.5:15:0.5 molar percent POPC:DOPS:NBD-DPPE). Controls with only buffer or liposomes were subtracted from the data. A correction for the slight fluorescence attenuation caused by light scattering by liposomes was determined by measuring the fluorescence of the amino acid Trp in the presence and absence of liposomes. % Total NBD Fluorescence 20 15 10 Tailless Tailless + 10 M CTH Tailless + 20 M melittin 5 Tailless + 40 M melittin 0 0 10 20 30 40 Time [min] Fig. S5. Melittin has only a weak ability to stimulate fusion of tailless ATL. The fusion activity of tailless ATL (reconstituted at a protein:lipid ratio of 1:1,000) was determined in the presence of different concentrations of melittin. For comparison, the curve for tailless ATL in the presence of CTH is shown. A CTH N-GGKLDDFATLLWEKFMRPIYHGCMEKGIH-C Reverse-CTH N-HIGKEMCGHYIPRMFKEWLLTAFDDLKGG-C B % Total NBD Fluorescence 25 20 15 Tailless Tailless + CTH Tailless + reverse-CTH 10 5 0 0 10 20 Time [min] 30 40 Fig. S6. A CTH peptide with the reversed sequence has a reduced ability to stimulate fusion of tailless ATL. (A) Prediction of helices for the wild-type peptide (CTH) and a peptide with the reverse sequence (reverse-CTH). (B) Fusion activity of the tailless ATL in the presence of 15 μM CTH or reverse-CTH peptide. Liu et al. www.pnas.org/cgi/content/short/1208385109 4 of 8 1.8 1.7 5-doxyl PC 12-doxyl PC 1.6 F0 /F 1.5 1.4 1.3 1.2 1.1 1.0 melittin N-BAR Sar1p reverseCTH L-Trp A Fluorescence (arbitrary units) B 1400 1200 1000 RPE-biotin PLs 800 Buffer 600 400 200 0 Fluorescence (arbitrary units) Fig. S7. Lipid interaction of amphipathic peptides used in Fig. 3 C and D and Fig. S6. The following peptides were added to liposomes containing or lacking phosphatidylcholine with doxyl groups at position 5 or 12 of the hydrocarbon chain: melittin, N-BAR helix (residues 1–24 of Rvs161p), Sar1p helix (residues 1–23 of Sar1p), and reverse CTH (a peptide with the reverse sequence of CTH; see Fig. S6). The quenching of the fluorescence of Trp residues in each of the peptides was measured and is expressed as F0/F (maximal fluorescence with doxyl-free liposomes divided by maximal fluorescence with doxyl-containing liposomes). A control using the amino acid Trp (L-Trp) instead of a peptide is shown for comparison. Results shown are the mean of three experiments. Error bars indicate SE. 350 300 250 100 50 0 SA-Cy5 - + + Detergent - - + Detergent WT + GTP WT + GTP + BDA 30 20 GTP or GDP WT WT + BDA WT + GDP WT + GDP + BDA 10 % Total Marina Blue Fluorescence % Total Marina Blue Fluorescence D 40 Buffer 150 RPE-biotin C SA-Cy5 PLs 200 - + + - - + 40 30 Tailless + CTH + GTP Tailless + GTP + CTH + BDA 20 GTP CTH 10 Tailless + CTH Tailless + CTH + BDA Tailless + GTP Tailless + GTP + BDA Tailless Tailless + BDA 0 0 0 5 10 20 15 Time [min] 25 30 0 5 10 20 15 Time [min] 25 30 Fig. S8. Encapsulation of content-mixing dyes into proteoliposomes reconstituted with wild-type ATL. (A) SA-Cy5 was added to proteoliposomes containing RPE-biotin in the absence or presence of detergent (1% Thesit). RPE-biotin fluorescence was measured in a SpectraMax M5 plate reader with an excitation wavelength of 540 nm and an emission wavelength of 575 nm. Quenching of RPE-biotin by SA-Cy5 occurred only when detergent was added to lyse the liposomes. (B) As in A, but RPE-biotin was added to proteoliposomes containing SA-Cy5 or buffer. FRET was measured by exciting RPE-biotin at a wavelength of 565 nm and detecting the fluorescence emission of SA-Cy5 at 675 nm. (C) Lipid mixing with wild-type ATL was measured in parallel with content mixing shown in Fig. 4A. The dequenching of the fluorescence of Marina Blue was monitored. (D) As in C, but with the content-mixing experiments shown in Fig. 4B. Liu et al. www.pnas.org/cgi/content/short/1208385109 5 of 8 1000 750 500 250 D A A5 11 88 74 L L4 Ta A4 ille ss 0 W T Fluorescence/OD600 1250 Fig. S9. Protein expression levels in the yeast complementation assay shown in Fig. 5. Wild-type human ATL1 or the indicated mutants were expressed under the SEY1 promoter in S. cerevisiae cells lacking Sey1p and Yop1p (sey1Δ yop1Δ cells). The expression levels were determined by immunoblotting with anti-ATL1 antibodies. Data shown are the mean of three experiments. Error bars indicate SE. A B 170 130 170 130 95 95 72 72 55 55 43 43 34 34 26 26 D D A WT A D A D ATL-Sac1p ATL-Sec61β D A WT C D 170 130 95 B100 Total A D ATL-LL S pH 11.0 P S A D cyt-TM1 P A cyt-TM2 TX-100 S P WT 72 55 cyt-TM1 43 cyt-TM2 34 ATL-LL 26 ATL-Sec61β D A WT D A L463A D A A449L D A L445A D A ATL-Sac1p G444L IB: anti-ATL Fig. S10. Reconstitution of TM mutants of ATL into liposomes. (A) Donor (D) and acceptor (A) vesicles, which were used for the fusion assays shown in Fig. 6B, were analyzed by SDS/PAGE and Coomassie blue staining. The vesicles contained wild-type or TM-replacement mutants of ATL. (B) As in A, but with TM insertion or deletion mutants shown in Fig. 6C. (C) As in A, but with TM point mutants shown in Fig. 6D. (D) Proteoliposomes containing WT or mutant ATL were incubated with a physiological buffer (B100), carbonate (pH 11.0), or 0.1% Triton X-100 (TX-100). The samples were centrifuged, and supernatants (S) and pellets (P) were analyzed by SDS/PAGE and immunoblotting with antibodies to Xenopus ATL, which cross-react with Drosophila ATL. Liu et al. www.pnas.org/cgi/content/short/1208385109 6 of 8 A % Total NBD Fluorescence 40 WT WT + CTH cyt-TM1 cyt-TM1 + CTH cyt-TM2 cyt-TM2 + CTH ATL-Sec61 ATL-Sec61 + CTH 30 20 10 0 -5 0 10 20 Time [min] 30 40 40 % Total NBD Fluorescence B WT WT + CTH cyt-TM1 cyt-TM1 + CTH cyt-TM2 cyt-TM2 + CTH ATL-Sec61 ATL-Sec61 + CTH 30 20 10 0 -5 0 10 20 Time [min] C 30 40 D 170 130 95 72 170 130 55 55 43 43 34 34 26 26 95 72 D A WT D A cyt-TM1 D A cyt-TM2 D A ATL-Sec61 D A WT D A cyt-TM1 D A cyt-TM2 D A ATL-Sec61 Fig. S11. Membrane fusion by wild-type ATL and TM mutants in the presence of CTH peptide. (A) Wild-type ATL or TM mutants lacking CT (cyt-TM1, cyt-TM2, and ATL-Sec61β) were reconstituted into liposomes at a protein:lipid molar ratio of 1:2,000. The membrane fusion assays were performed in the absence or presence of CTH peptide. (B) As in A, but the proteins were reconstituted at a protein:lipid molar ratio of 1:400. Note that the cyt-TM2 mutant showed some low fusion activity in the presence of CTH peptide. (C) The donor (D) and acceptor (A) vesicles used in A were analyzed by SDS/PAGE and Coomassie blue staining. (D) As in C, but with vesicles used in B. Liu et al. www.pnas.org/cgi/content/short/1208385109 7 of 8 TM1 A DmATL TM2 AA LL L LA LL ALL AL LA A A AA 423 ...PAVYFACAVIMYILSGIFGLVGLY TFAN FCNLVMGVALLTLALWAYIRY... HsATL1 448 ...PATLFVVIFITYVIAGVTGFIGLD IIAS LCNMIMGLTLITLCTWAYIRY... HsATL2 475 ...PATLFAVMFAMYIISGLTGFIGLN SIAV LCNLVMGLALIFLCTWAYVKY... HsATL3 467 ...PAVLFTGIVALYIASGLTGFIGLE VVAQ LFNCMVGLLLIALLTWGYIRY... LlATL 471 ...PAVLVTFMIVDYVMQEFFQLVGLD TIAG LFSAALCVAVVSLSIWAYSRY... XtATL1 448 ...PATLFVVIFITYVLAAVTGFIGLD IIAS LCNMIMGLTLITLCTWAYIRY... MmATL1 448 ...PATLFVVIFITYVIAGVTGFIGLD IIAS LCNMIMGLTLITLCTWAYIRY... SmATL 426 ...PAVLAVVLLIFHIVTGISEFIGLS MVSG ILALPFYIALVSLFTWLFLSY... CqATL 424 ...PAVYFAIAVVMYIFSGIFGLVGLY TFAN FANLIMGVALLTLATWAYIRY... AaATL 426 ...PAVYFAIAVVMYIFSGIFGLVGLY TFAN FANLIMGIALLTLATWAYIRY... BmATL 471 ...PAVLVTFMIVDYVLQEFFQLIGLD IIAG LFSAALCIAVVSLGIWAYSRY... Fig. S12. Sequence alignment of TMs of ATLs from various species and fusion activity of tested TM mutants: Drosophila melanogaster ATL (DmATL), Homo sapiens ATL1 (HsATL1), Homo sapiens ATL2 (HsATL2), Homo sapiens ATL3 (HsATL3), Loa loa ATL (LlATL), Xenopus tropicalis ATL1 (XtATL1), Mus musculus ATL1 (MmATL1), Schistosoma mansoni ATL (SmATL), Culex quinquefasciatus ATL (CqATL), Aedes aegypti ATL (AaATL), Brugia malayi ATL (BmATL). The indicated DmATL residues were changed to the residues shown above the DmATL sequence. The fusion activity of the purified mutants was determined and classified into wild type-like (blue) and reduced activity (red). 1 75KD 43KD 158KD 669KD 440KD 2 5 7 3 4 6 8 9 10 11 12 13 WT (1% SDS) WT (1% Triton X-100) WT (1% Digitonin) WT (1% Digitonin+EDTA) IB: anti-ATL Fig. S13. ATL forms nucleotide-independent oligomers. Proteoliposomes containing wild-type ATL were solubilized in 1% SDS, 1% Triton X-100, 1% digitonin, or 1% digitonin and EDTA, and the extracts were subjected to sucrose gradient centrifugation. Fractions were analyzed by SDS/PAGE and immunoblotting with antibodies to Xenopus ATL. Note that ATL oligomers are dissociated by both SDS and Triton X-100. Molecular mass standards in the sucrose gradient are indicated. Liu et al. www.pnas.org/cgi/content/short/1208385109 8 of 8