Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2012 Aug 7;109(32):E2146-54.
doi: 10.1073/pnas.1208385109. Epub 2012 Jul 16.

Lipid Interaction of the C Terminus and Association of the Transmembrane Segments Facilitate Atlastin-Mediated Homotypic Endoplasmic Reticulum Fusion

Affiliations
Free PMC article
Comparative Study

Lipid Interaction of the C Terminus and Association of the Transmembrane Segments Facilitate Atlastin-Mediated Homotypic Endoplasmic Reticulum Fusion

Tina Y Liu et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

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 C-terminal 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.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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.
Fig. 2.
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. 3.
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.
Fig. 4.
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.
Fig. 5.
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.)
Fig. 6.
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.
Fig. 7.
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 Myc- and Flag-tagged dmATL constructs. A contaminating Ig band from the antibody-conjugated matrix used in the IP is indicated by an asterisk.
Fig. P1.
Fig. P1.
A model for ATL-mediated homotypic ER fusion. The membrane-bound GTPase ATL is postulated to mediate the fusion of ER membranes in a process involving the following steps. (A) ATL molecules in the same membrane associate in a nucleotide-independent manner through their transmembrane segments. (B) These complexes bind GTP and interact with similarly assembled ATL molecules in another membrane. (C) GTP hydrolysis and phosphate (Pi) release triggers a large conformational change that pulls the two membranes toward each other. The amphipathic helix in the CT of ATL, shown as magenta and yellow circles, facilitates fusion between the approaching membranes by perturbing the lipid bilayers. (D and E) Once fusion has occurred, GDP release allows nucleotide-dependent ATL dimers to dissociate and start a new round of fusion. 3HB, three-helix bundle; G, GTPase domain.

Similar articles

See all similar articles

Cited by 50 articles

See all "Cited by" articles

Publication types

MeSH terms

Feedback