Download Lipid interaction of the C terminus and association of the

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.
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membrane perturbation content mixing organelle shaping
organelle remodeling hereditary spastic paraplegia
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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]
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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.
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Liu et al.
PNAS PLUS
Fluorescence Microscopy in Yeast. 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
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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
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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
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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
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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
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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
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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
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