(A) Detergent extracts from mouse brain or spinal cord were incubated with peptide-specific antibodies to ATL1. Where indicated, antibodies were preincubated with the antigenic peptide. Immunoprecipitated proteins were analyzed by SDS-PAGE and Coomassie staining. Rtn4a was identified by mass spectrometry (5 distinct peptides covering 74 of the 1163 amino acids). An asterisk (*) indicates the position of the IgG heavy chain. M, molecular mass standards (in kDa).
(B) Proteins immunoprecipitated (IP) with ATL1 antibodies or control IgG were analyzed by immunoblotting (IB) with Rtn4a antibodies. Lanes 1 and 2 (loads) show 10% of the starting material used for immunoprecipitation.
(C) Proteins immunoprecipitated with Rtn4a antibodies or control IgG were immunoblotted with ATL1 antibodies. Lanes 1 and 2 (loads) contain 10% of the starting material used for the immunoprecipitations.

(A) Myc-ATL1 and HA-Rtn3c were expressed either individually or together in COS-7 cells. Digitonin-extracts were used for immunoprecipitation (IP) with anti-HA, anti-Myc, or anti-FLAG antibodies. Ten percent of the starting material (load) and of the material not bound to the antibodies (unbound), as well as the precipitates were analyzed by SDS-PAGE and immunoblotting (IB) with anti-HA or anti-Myc antibodies. As a control, the blot was also probed with antibodies to the integral ER membrane protein TRAPalpha.
(B) Myc-ATL1 or Myc-ATL1 K80A was expressed together with HA-tagged DP1 (HA-DP1) in COS-7 cells. Immunoprecipitation was performed as in (A).
(C) As in (B), but with COS-7 cells expressing Myc-ATL1 and the HA-tagged reticulon homology domain of Rtn4a (HA-Rtn4HD).
(D) As in (B), but with cells expressing Myc-ATL1 and the HA-tagged N-terminal, cytoplasmic segment of Rtn4a (HA-cytRtn4a).
(E) As in (B), but with cells expressing the transmembrane domain of ATL1 (Myc-ATL1TM) and HA-Rtn3c.

(A) Myc-ATL1 was expressed in COS-7 cells. Its localization was identified with anti-Myc antibodies (green) and compared to that of an endogenous luminal ER protein, calreticulin (red) using indirect immunofluorescence and confocal microscopy. Lower panels show an enlargement of the boxed region centered on ER sheets. Bar, 10 μm.
(B) As in (A), but a cell is shown with particularly high expression of Myc-ATL1. ER sheets are aberrant.
(C) As in (A), but with cells expressing the GTP-binding mutant Myc-ATL1 K80A. Shown is a cell with an unbranched ER tubule phenotype.
(D) As in (A), but with cells expressing a Myc-ATL1 construct that contains only the N-terminal cytoplasmic domain (Myc-cytATL1). Cells also expressed a RFP-tagged version of the ER protein Sec61β and were then stained with Myc- and calreticulin-antibodies. Shown is a cell with an unbranched ER tubule phenotype.
(E) Myc-ATL1, Myc-ATL1 K80A, Myc-ATL1 R217Q, GFP-Sec61β, or GFP-Rtn4a were expressed in COS-7 cells and the percentage of cells with normal ER, with aberrant sheet-like ER structures, or with unbranched tubules was determined. Means ± standard errors were calculated from three different samples of each transfection (200-500 cells per sample).

(A) HeLa cells were transfected with siRNA oligonucleotides directed against ATL2 and ATL3 (ATL DKD) or with control siRNA. ATL2 and ATL3 protein levels were then determined by immunoblotting. PLCγ levels were monitored as a loading control.
(B) HeLa cells depleted or not depleted for ATL2 and ATL3 were transfected with GFP-Sec61β and visualized by fluorescence confocal microscopy. Bar, 10 μm.
(C) The number of cells displaying the “unbranched tubule” phenotype (lower panel in (B)) was quantitated as a percentage relative to the total number of cells (means ± SD from three different experiments, with 100 cells per experiment). P<0.02.

Membranes from Xenopus eggs were preincubated for 60 min with increasing concentrations of affinity-purified, pan-ATL antibodies or with control IgG. GTP was then added, and network formation was visualized by fluorescence microscopy after staining with octadecyl rhodamine. Bar, 20 μm.

(A) Predicted membrane topology of Sey1p.
(B) Signature motifs of the GTPase domains of the Sey1p/atlastin family. G1, G2, and G3 motifs shared with dynamin-1 are shown in orange, and the divergent G4 motifs of the Sey1p/atlastin family and of dynamin-1 are shown in yellow and green, respectively. The atlastins, GBP1, and dynamin-1 are human forms, Sey1p is from S. cerevisiae (S. c.), and RHD3 is from Arabidopsis thaliana (A. t.). Residue numbers shown are for atlastin-1 and Sey1p.
(C) Sey1p-GFP was expressed under the endogenous promoter from a CEN plasmid together with RFP targeted to the ER lumen (ss-RFP-HDEL). Proteins were visualized by fluorescence microscopy, focusing either at the center or periphery of the cells. Bar, 1 μm.
(D) HA-Sey1p and Yop1p-FLAG were co-expressed individually or together under their endogenous promoters on CEN plasmids in a sey1Δyop1Δ strain. Proteins were immunoprecipitated (IP) from cell lysates using HA or FLAG antibodies, separated by SDS-PAGE, and analyzed by immunoblotting (IB) with HA or FLAG antibodies. Similar experiments were also performed with a Sey1p mutant (Sey1p K50A) that is defective in GTP binding.
(E) As in (D), except that HA-Sey1p and Myc-tagged Rtn1p (Rtn1p-Myc) were expressed individually or together in a sey1Δrtn1Δ strain, and Myc-epitope antibodies were used.

(A) A GFP-fusion of the ER protein Sec63p was expressed in cells lacking Sey1p (sey1Δ). The localization of the protein was determined by fluorescence microscopy, focusing the microscope either at the center or the periphery of the cell. Bar, 1 μm.
(B) As in (A), but with cells lacking both Sey1p and Rtn1p (sey1Δrtn1Δ).
(C) As in (A), but with cells lacking both Sey1p and Yop1p (sey1Δyop1Δ).
(D) As in (C), but with cells expressing wild-type Sey1p from a CEN plasmid.
(E) As in (C), but with cells expressing a GTP-binding mutant of Sey1p (Sey1p K50A) from a CEN plasmid.
(F) The percentage of cells with abnormal ER was determined from 20-40 cells per mutant. The small percentage of cells with abnormal ER in (D) likely reflects loss of the plasmid.