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First published online 14 April 2008
doi: 10.1242/jcs.028530

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SNX18 is an SNX9 paralog that acts as a membrane tubulator in AP-1-positive endosomal trafficking

Department of Medical Biochemistry and Biophysics, Umeå University, S-901 87 Umeå, Sweden

View larger version (41K): [in this window] [in a new window]   Fig. 1. The SNX9 protein family. (A) Domain organization of SNX9, SNX18 and SNX30. For the explanation of domain designations, see text. The numbers refer to the last amino acid in each domain in human proteins. (B) Pairwise sequence identities of the different domains. Individual domain sequences were aligned with ClustalW allowing for default gap insertions. (C) Immunoblotting of mouse tissue extracts with antibodies against the three paralogs and with anti-amphiphysin-1 antibodies (Amph1). The same amount of protein was loaded in each lane. The relative molecular mass of the respective protein compared to protein standards is indicated to the right.

View larger version (63K): [in this window] [in a new window]   Fig. 2. Characterization of SNX9, SNX18 and SNX30 proteins in cells. (A) HeLa-cell cytosol was immunoprecipitated with antisera against the three paralogs, and with pre-immune serum, and analyzed with SDS-PAGE. The gel was stained with Coomassie. Bands corresponding to IgG and to immunoprecipitated SNX9, SNX18 and SNX30 are indicated. The identity of SNX18 and SNX30 was determined by MALDI-TOF. Arrows show the positions of dynamin 2 (upper) and aldolase (lower). Inset below shows an immunoblotting analysis with respective antibody of immunoprecipitates from a total HeLa-cell detergent extract. Note that individual paralogs do not form complexes with other family members. (B) Epifluorescent micrograph of a HeLa cell transfected with myc-SNX30 and stained with mouse anti-myc, rabbit anti-SNX9 and chicken anti-SNX18. Scale bar: 10 µm. Enlarged pictures show no overlap in localization of the three proteins. (C) A membrane fraction from HeLa cells was separated by density equilibrium centrifugation, and collected fractions were analyzed for density (triangles) and total protein amount (circles). The distribution of the indicated proteins was analyzed by immunoblotting.

View larger version (57K): [in this window] [in a new window]   Fig. 3. SNX9, SNX18 and SNX30 interact and colocalize with dynamin 2. (A) GST or GST-fusion proteins with SH3-domains from SNX9, SNX18 or SNX30 were incubated with (+) or without (–) cytosol from HeLa cells. After washings, bound protein was analyzed by SDS-PAGE and the gel was stained with Coomassie. The identity of dynamin 2 (Dyn2) was verified with immunoblotting. (B) The PRD of dynamin 2, containing wild-type (wt) or mutated (mut1-4) sequences, was purified and used in pull-downs with GST or GST-fusion proteins containing SH3-domains from SNX9, SNX18, SNX30 or amphiphysin 2 (Amph2), as described in the Materials and Methods. Bound protein, together with total protein (Input), was analyzed by SDS-PAGE and the gels were stained with Coomassie. The sequence of the C-terminal part of the PRD with indicated type I and type II SH3-binding motifs is shown below. Modified amino acids in mutated PRDs are highlighted. (C) Epifluorescent micrograph of HeLa cells expressing myc-tagged SNX9 (a) or myc-tagged SNX18 (b) and counterstained for dynamin 2 after wash-out of cytosolic components as described in the Materials and Methods. Magnifications of the boxed areas together with illustrations of the specific structures labeled with anti-myc and anti-dynamin-2 are shown to the right. Scale bar: 10 µm.

View larger version (102K): [in this window] [in a new window]   Fig. 4. Membrane-binding and -tubulation by PX-BAR domains from SNX9, SNX18 and SNX30. (A) Epifluorescent micrographs of HeLa cells expressing myc-tagged PX-BAR domains from SNX9, SNX18 and SNX30. Scale bar: 10 µm. Note the long, myc-stained tubules formed by all three PX-BAR constructs. (B) Electron micrograph of membrane tubes generated by incubation of liposomes made from brain-derived lipids together with 20 µM of purified SNX18-PX-BAR protein. Inset shows 2.5x magnification of the indicated area. (C) Phosphoinositide-binding specificity of SNX18-PX-BAR analyzed by liposome co-sedimentation. SNX18-PX-BAR was incubated with liposomes made from brain-derived lipids (Brain), or with liposomes with defined lipid composition without (PS) or with indicated phosphoinositides (see Materials and Methods). After centrifugation, pellet (P) and supernatant (S) fractions were analyzed by SDS-PAGE and Coomassie-stained bands were quantitated by densitometry. (Below) The bars show the means from two experiments, with the maximum value indicated for each set. (D) Epifluorescent micrographs showing HeLa cells expressing myc-tagged SNX18-PX-BAR treated with ionomycin or wortmannin as indicated. Note the loss of membrane tubulation after ionomycin treatment. Scale bar: 10 µm. All PX-BAR constructs contained the contiguous Y domains (see Fig. 1A).

View larger version (36K): [in this window] [in a new window]   Fig. 5. SNX18 colocalizes and interacts with AP-1. (A) HeLa cells were stained for endogenous SNX18 and AP-1, and visualized by epifluorescence. Scale bar: 10 µm. The indicated boxed area was magnified and arrows highlight extensive colocalization in the periphery. (B) Pull-down experiment with GST and GST-fusion proteins containing ear domains of the mammalian AP complexes. Fusion proteins bound to beads were incubated with His-tagged LC-region from SNX18, and bound and unbound SNX18-LC was analyzed by SDS-PAGE and filter-blotted with His-probe (Pierce). The Coomassie-stained gel shows the fusion proteins. (C) Pull-down experiment with GST and GST-fusion proteins containing ear domains of 1 or the related subunit 2, or the GAE domains of GGA1 or GGA2. Pull-down was performed as in B. (D) Demonstration of a specific 1-binding site in SNX18. The alignment shows a conserved acidic patch in the LC region of SNX18. Black shade indicates conserved tryptophans and acidic residues, and gray shade indicates flanking amino acids with conserved properties in at least four out of five species. Bead-bound GST and GST-1-ear were incubated with wild-type (wt) or mutated (W154S and W158S, arrows in the alignment) His-SNX18-LC, and pull-down was performed as in B.

View larger version (88K): [in this window] [in a new window]   Fig. 6. SNX18 is part of an endosomal AP-1- and PACS1-positive budding machinery. (A) Fluorescent micrograph of a HeLa cell treated with BFA and co-stained for endogenous SNX18 and AP-1. Magnification of the boxed area is shown to the right; arrows indicate colocalized puncta. (B) The graph shows the degree of colocalization between SNX18 and AP-1 in untreated and BFA-treated HeLa cells. The bars show the percentage of SNX18-positive puncta that colocalized with AP-1, as means (s.e.m.) from a total of 45 5x5-µm squares placed over 25 randomly selected cells. Student's t-test showed P<0.01 between untreated and treated cells. (C) Lysates from BFA-treated or untreated HeLa cells were immunoprecipitated (IP) with antisera against -adaptin or without primary antibody (Control), and analyzed by immunoblotting (Blot) using antisera against SNX18 and -adaptin. Co-immunoprecipitation increased after BFA-treatment. (D) Epifluorescent micrograph showing a HeLa cell overexpressing myc-tagged SNX18 (green) together with untransfected cells, stained for AP-1 (red) after washout of cytosolic components. For clarity, the cell borders are outlined as broken lines. Note the disappearance of peripheral AP-1 puncta in the SNX18-overexpressing cell. (E) Epifluorescent micrograph showing a HeLa cell stained for endogenous SNX18 and PACS1. Arrows in the magnified merged inset illustrate puncta in which the proteins co-localized. Scale bars: 10 µm.

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