Atlastin GTPases are required for Golgi apparatus and ER morphogenesis

Abstract

The hereditary spastic paraplegias (SPG1-33) comprise a cluster of inherited neurological disorders characterized principally by lower extremity spasticity and weakness due to a length-dependent, retrograde axonopathy of corticospinal motor neurons. Mutations in the gene encoding the large oligomeric GTPase atlastin-1 are responsible for SPG3A, a common autosomal dominant hereditary spastic paraplegia. Here we describe a family of human GTPases, atlastin-2 and -3 that are closely related to atlastin-1. Interestingly, while atlastin-1 is predominantly localized to vesicular tubular complexes and cis-Golgi cisternae, mostly in brain, atlastin-2 and -3 are localized to the endoplasmic reticulum (ER) and are most enriched in other tissues. Knockdown of atlastin-2 and -3 levels in HeLa cells using siRNA (small interfering RNA) causes disruption of Golgi morphology, and these Golgi structures remain sensitive to brefeldin A treatment. Interestingly, expression of SPG3A mutant or dominant-negative atlastin proteins lacking GTPase activity causes prominent inhibition of ER reticularization, suggesting a role for atlastin GTPases in the formation of three-way junctions in the ER. However, secretory pathway trafficking as assessed using vesicular stomatitis virus G protein fused to green fluorescent protein (VSVG-GFP) as a reporter was essentially normal in both knockdown and dominant-negative overexpression conditions for all atlastins. Thus, the atlastin family of GTPases functions prominently in both ER and Golgi morphogenesis, but they do not appear to be required generally for anterograde ER-to-Golgi trafficking. Abnormal morphogenesis of the ER and Golgi resulting from mutations in atlastin-1 may ultimately underlie SPG3A by interfering with proper membrane distribution or polarity of the long corticospinal motor neurons.

Figure 1.

(A) Atlastin protein phylogeny. Species names and GenBank accession numbers are to the right. In higher species, there are three atlastin family members: atlastin-1 (green), atlastin-2 (yellow), and atlastin-3 (lavender), while a number of lower species have only one atlastin family member (top). The tree was constructed using ClustalW (v. 1.4) and MacVector 7.2. (B) Anti-peptide antibodies were raised against divergent regions of human atlastin-1, -2 and -3 as well as to a region common to all three (AT1-3) and used for immunoblotting extracts from cells overexpressing the indicated atlastins. Though atlastin proteins are expressed endogenously in COS7 cells, the endogenous proteins are not seen in immunoblots at these short exposure times; however, they are seen at longer exposures (unpublished data). Anti-atlastin-2ab (αAT2ab) is directed against a sequence present in both known C-terminal splice variants of atlastin-2: -2a and -2b. (C) Extracts were prepared from untransfected (Untrans) COS7 cells and cells transfected with AT1, AT2b, or AT3 (Trans) and then immunoblotted with the corresponding antibodies; sizes of the endogenous proteins were assessed by immunoblotting untransfected human 293 cell extracts. For atlastin-3, two different translation start sites were examined, and the shorter form (Trans-S) closely matched the size of the endogenous protein in 293 cells (arrowhead). An asterisk indicates a cross-reactive band. (D and E) Extracts from the indicated adult human tissues (D) and immortalized and primary human cell lines (E) were probed with the indicated anti-atlastin antibodies. Calnexin levels were monitored in (E) as a control for protein loading (Sk muscle, skeletal muscle; Sm muscle, smooth muscle).

Figure 2.

Membrane topology of atlastin proteins. (A) Triton X-114 phase partitioning. Homogenates from COS7 cells transfected with atlastin-1, -2, or -3 were partitioned with Triton X-114 and aliquots of the starting material (Homog) as well as aqueous and detergent (Deterg) phases were immunoblotted with the indicated antibodies. The cytoplasmic protein PLCγ was monitored as a control for phase partitioning. (B) Hydrophobic domains of the atlastins are required for membrane association. Post-nuclear supernatants from COS7 cells expressing either Myc-tagged full-length (FL) or the indicated Myc-tagged deletion constructs for atlastin-1, -2 and -3 were fractionated into membrane (Memb) and soluble (Sol) fractions and immunoblotted with anti-Myc antibodies. Deletion of both predicted transmembrane domains (ΔH-H) markedly reduced membrane association, while C-terminal fragments containing these domains (H-H) were sufficient for membrane association. ΔH-H: atlastin-1 (1–447); atlastin-2a (1–474); atlastin-3 (1–417). H-H: atlastin-1 (448–558); atlastin-2a (475–579); atlastin-3 (418–515). Amino acid residues are numbered as in Supplementary Material, Fig. S1. (C) Protease protection assays. Microsomal (P3) fractions from cells transfected with N-terminally Myc-tagged atlastin-1, -2, or -3 were treated with proteinase K (Prot K) as indicated and then immunoblotted for Myc (αN-term), the corresponding C-terminal atlastin antibodies (αC-term), calnexin, and Grp78. A small portion of the transmembrane calnexin protein is oriented to the cytoplasm and thus it is partially degraded, while Grp78 is wholly within the ER lumen and protected from proteolysis. (D) Schematic structure of the proposed membrane topology for the atlastin proteins (Cyto, cytoplasm).

Figure 3.

Atlastins form homomeric protein complexes in cells. (A) Chemical cross-linking. Extracts from COS7 cells expressing Myc-tagged atlastin-1 (AT1), -2b (AT2), and -3 (AT3) were cross-linked with 0.25 mm bis(sulfosuccinimidyl)suberate (BS³) and immunoblotted with anti-Myc antibodies. Asterisks denote probable dimeric and tetrameric forms, while an arrowhead identifies a putative trimer. Migrations of molecular mass standards (in kDa) are indicated in the left. (B) Extracts from COS7 cells expressing atlastin-1, -2 and -3 were subjected to gel-exclusion FPLC. Aliquots of each collected fraction were immunoblotted with anti-Myc antibodies. Elution peaks for marker proteins (in kDa) are shown across the top. Fraction numbers are indicated along the bottom. (C) Co-immunoprecipitation of endogenous atlastin proteins. Triton X-100 solubilized 293 cell extracts were immunoprecipitated (IP) with antibodies raised in guinea pigs or goats against atlastin-1 or -2b, respectively, or else control IgG, and then immunoblotted for atlastin-1, -2b, or -3 using antibodies raised in rabbits. Input represents 20% of the starting material.

Figure 4.

Atlastin-2 and -3 localize to the ER. (A) HeLa cells were co-immunostained for endogenous atlastin-2 or -3 (green) and the cis/medial-Golgi marker GM130 (red). Merged images are to the right. (B) HeLa cells were transfected with VSVG-GFP (green) and fixed during the 40°C incubation to reveal the ER. Cells were then immunostained for endogenous proteins using antibodies specific for atlastin-2 and -3 (red). Merged images are at the right. Bars, 10 µm.

Figure 5.

Atlastin-2 and -3 localize to ER membranes and along microtubules by immunogold electron microscopy. Electron microscopic analysis of HeLa cells immunostained for endogenous atlastin-2 (top and middle panels) or atlastin-3 (bottom panel) reveals gold particles not only at ER and VTCs/cis-Golgi (top and bottom panels), but also decorating membranes along microtubules (middle panel) (G, Golgi apparatus; MT, microtubules). Bars, 100 nm.

Figure 6.

siRNA-mediated knockdown of atlastin-2 and -3 disrupts the Golgi apparatus. (A) Lysates from HeLa cells transfected with three different siRNAs specific for atlastin-2 (AT2) or else control siRNA were immunoblotted for endogenous atlastin-2 and -3 at 72 h post-transfection (left panels). Equal protein loading was monitored by immunoblotting for PLCγ. There is no upregulation of atlastin-3 protein expression upon knockdown of atlastin-2. Lysates from HeLa cells transfected with three different siRNAs specific for atlastin-3 (AT3) or else control siRNA were immunoblotted for atlastin-2 and -3, with PLCγ levels monitored as a loading control (right panels). Atlastin-2 expression is not upregulated upon knockdown of atlastin-3. An asterisk in atlastin-3 immunoblots identifies a cross-reactive band. (B) Lysates from HeLa cells transfected with both AT2 siRNA #1 and AT3 siRNA #3 or else control siRNA were immunoblotted for atlastin-2 and -3, with PLCγ levels monitored as a control for protein loading. (C) Graphical representation of percentage of cells with disrupted Golgi morphology in atlastin-2, -3, or double knockdown (DKD) conditions as compared with control siRNA-transfected cells (n = 3; 100 cells per condition). *P < 0.01; **P < 0.001. (D) HeLa cells transfected with control siRNA or atlastin-2 siRNA #1 were co-immunostained for atlastin-2 and GM130 (left panels) 72 h after transfection. HeLa cells were also transfected with control siRNA or atlastin-3 siRNA #3 and co-immunostained for atlastin-3 and GM130 (right panels). Boxed areas are enlarged in the insets to show fragmentation or tubular Golgi extending beyond the perinuclear region. Bars, 10 µm.

Figure 7.

Tubular Golgi structures in atlastin siRNA cells are sensitive to brefeldin A (BFA). HeLa cells transfected with either control or atlastin-2 siRNAs were re-transfected with an YFP-Golgi expression construct and then treated with BFA 24 h later. Live cell images were acquired during the BFA treatment to determine whether the tubular structures seen in atlastin-2 siRNA cells were sensitive to BFA. As shown, the distribution of YFP-Golgi changes when comparing cells before BFA treatment (upper panels) to after treatment (lower panels). Similar results were also seen in cells depleted of atlastin-3 using siRNA (unpublished data). See also Supplementary Material, Videos 1–3. Bars, 10 µm.

Figure 8.

VSVG trafficking is not impaired in cells lacking both atlastin-2 and -3. HeLa cells were transfected with control siRNA (left panels), or with both atlastin-2 and -3 siRNAs (DKD (double knockdown); right panels), and both cell groups were then re-transfected with ts045 VSVG-GFP. The VSVG can be seen at perinuclear pre-Golgi and/or Golgi structures in both cell groups 60 min after moving to a permissive temperature. VSVG trafficked to the plasma membrane by 180 min in control cells as well as atlastin-2 and -3 DKD cells. Bars, 10 µm.

Figure 9.

Dominant-negative atlastin proteins prominently disrupt ER morphology. HeLa cells expressing Myc-tagged atlastin-1, -2 or -3 (upper panels) exhibit a typical reticular ER morphology, as revealed by co-labeling with VSVG-GFP at 40°C. However, expression of Myc-tagged SPG3A atlastin-1 mutant R217Q and the equivalent mutations atlastin-2^R244Q or atlastin-3^R187Q result in a more tubular, elongated ER morphology with much less branching (middle panels). This profound effect on ER morphology was also seen to the same, if not greater, extent upon expression of Myc-tagged dominant-negative mutations atlastin-1^K80A, atlastin-2^K107A, or atlastin-3^K47A (bottom panels). Bars, 10 µm.

Figure 10.

Dominant-negative atlastin proteins disrupt Golgi morphology but do not alter anterograde secretory trafficking. (A) Expression of Myc-tagged dominant-negative (DN) atlastin-1, -2 or -3 (left panels) in HeLa cells results in Golgi fragmentation in a majority of transfected cells (right panels), as revealed by co-immunostaining for Myc-epitope and GM130. An arrow identifies a morphologically normal Golgi apparatus in an untransfected cell. (B) Graphical representation of percentage of cells with disrupted Golgi morphology in DN atlastin-1, -2, or -3 expressing cells versus control cells (n = 3; 100 cells per condition). *P < 0.01; **P < 0.001. (C) HeLa cells either singly transfected with ts045 VSVG-GFP or else co-transfected with VSVG-GFP and Myc-tagged atlastin-3 DN exhibit VSVG-GFP within ER before being moved to the permissive temperature (left panels). 180 min after being moved to the permissive temperature, cells from both groups are able to traffic VSVG to the plasma membrane despite the abnormal ER morphology (right panels). Similar results were seen when using atlastin-1 and -2 DN constructs (unpublished data). Bars, 10 µm.

Figure 11.

Interactions of atlastin family members with the SPG4 protein spastin. (A and B) Yeast two-hybrid tests showing interactions of the spastin bait construct with full-length atlastins (A) and the indicated atlastin-1 deletion constructs, with residues 1-558 representing the full-length atlastin-1 protein (B), as revealed by growth selection on -His/Leu/Trp plates. The -Leu/Trp panel demonstrates similar transformation efficiencies for each reaction. Sequential 10-fold dilutions of yeast are shown for all panels. Strength of interaction was also assessed semi-quantitatively by determining the time for colonies to turn blue in X-gal filter lift assays: +++, <30 min; ++, 30–60 min, +, 60–120 min; and −, no significant activity.