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First published online 1 June 2004
doi: 10.1242/jcs.01170

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Mammalian class E Vps proteins, SBP1 and mVps2/CHMP2A, interact with and regulate the function of an AAA-ATPase SKD1/Vps4B

¹ Division of Pharmaceutical Cell Biology, Kyushu University Graduate School of Pharmaceutical Sciences, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
² National Institute of Genetics, Department of Cell Genetics, Mishima, 411-8540, Japan
³ Laboratory for Genome Exploration Research Group, RIKEN Genomic Sciences Center (GSC), Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan
⁴ Department of Biochemistry, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo, 113-8421, Japan

View larger version (55K): [in a new window]   Fig. 1. (A) CLUSTAL W sequence alignment of mouse SBP1 and Vta1p, a putative yeast homologue. (B) CLUSTAL W sequence alignment of mouse and yeast Vps2p. (C) Domain structures of SBP1, Vta1p, mVps2 and Vps2p. Predicted coiled-coil domains are marked with coils. The dotted line through the center of molecules divides the proteins into their positively charged N-terminal and negatively charged C-terminal domains. Molecular masses are given on the right, as calculated from their amino acid composition and determined by SDS-PAGE. Data of Vps2p was described previously (Babst et al., 2002a). N.D. is not determined. (D) Western blotting analysis of SBP1 in mouse tissues. Total extracts from various mouse tissues (50 µg of protein) were run on 10% SDS-PAGE and analyzed by western blotting with affinity purified anti-SBP1 polyclonal antibody. (E) SDS-PAGE analyses of bacterially expressed mVps2. GST-mVps2 was purified with glutathione-beads (lane 1) and subjected to thrombin cleavage on the beads (lane 2). Subsequently, the mVps2 portion was recovered by sedimentation of the beads with centrifugation (lane 3). Coomassie Blue staining revealed that both GST-mVps2 (60 kDa) and thrombin-cleaved mVps2 (35 kDa) migrated slower than expected from their molecular masses (51.1 and 25.1 kDa, respectively). (F) Northern blotting analysis of SBP1. Full-length SBP1 cDNA was labeled with 32P by random priming and hybridized to multiple tissue northern blots (Clontech) under high stringency conditions. 2 µg of poly(A) + RNA from various mouse tissues was loaded in each lane. (G) Northern blotting analysis of mVps2. As for SBP1, mVps2 mRNA was detected on Multiple Tissue northern blots (Clontech) using full-length mVps2 cDNA as probe.

View larger version (35K): [in a new window]   Fig. 2. In vitro binding analyses of SBP1 and SKD1 (A) Two hybrid binding analyses. Rectangles represent the protein structure of SBP1 (309 amino acids) and the five clones that were obtained from yeast two-hybrid screening. Mav203 cells transformed with pDBLeu-SKD1 and pPC86-SBP1 (clone 1-5) were plated on SC His+ Trp- Leu- (1, 2), SC His- Trp- Leu- (3) and SC His- Trp- Leu- Ura- (4) plates. An X-gal assay was performed on the nitrocellulose filter (2). Mav203 cells, transformed with the two plasmids supplied with the kit (pPC97-Fos and pPC86-Jun) as a positive control (P.C.), or with the empty vector pPC86 and pDBLeu-SKD1 as a negative control (pPC86) are indicated. (B) SPR spectroscopy. Indicated concentrations (30, 60, and 150 µg/ml) of His-thioredoxin-SBP1 (full length) in binding buffer were injected over the immobilized GST-SKD1 on a CM5 sensor chip. An arrowhead and arrow indicate the sample and dissociation buffer injection points, respectively. (C) GST pull-down assay. GST or GST-SKD1 was immobilized on glutathione-sepharose prior to incubation with purified His-thioredoxin-SBP1s [full length (1-309), N (198-309), and C (1-129)]. Bound proteins were eluted with SDS sample buffer and processed for immunoblotting with anti-SBP1 antibody.

View larger version (29K): [in a new window]   Fig. 3. Effects of SKD1(E235Q) on the subcellular localization and oligomer formation of SBP1. (A) Post nuclear supernatant prepared from control and GFPSKD1(E235Q) adenovirus-infected rat 3Y1 fibroblast cells were fractionated into cytosol (sup.) and membrane (ppt.) by centrifugation at 105,000 g for 60 minutes. Comparable amounts of each fraction were processed for immunoblotting with anti-SKD1 and anti-SBP1 antibodies. (B) A cell lysate prepared from HeLa cells with binding buffer was fractionated by size exclusion chromatography on a Superdex 200 column. The collected fractions were processed for immunoblotting with anti-SKD1 and anti-SBP1 antibodies. Positions of molecular mass standards are indicated by arrows. (C) A cell lysate prepared from GFP-SKD1(E235Q) adenovirus-infected HeLa cells was fractionated and analyzed as described in B. (D) Chemical cross-linking of SBP1. NIH3T3 cell lysates were incubated with non-cleavable cross-linker DSS (0, 10, 100 and 1000 µM) for 60 minutes and then processed for immunoblotting with anti-SBP1 antibody.

View larger version (79K): [in a new window]   Fig. 4. Overexpression of SKD1(E235Q) specifically changes the distribution of endogenous SBP1. (A) HeLa cells transfected with GFP-SKD1(E235Q) were subjected to immunofluorescence analysis using anti-SBP1 antibody. GFP and Cy3 fluorescence signals obtained from GFP-SKD1(E235Q) and Cy3-conjugated secondary antibody were labeled with green and red pseudo color, respectively. The transfected cells are indicated by asterisks in the red color image. Notice, the transfected cells showed a redistribution of SBP1 to the aberrant membranous structures, E235Q compartments, while the non-transfected cells showed a cytoplasmic distribution of SBP1. (B) HeLa cells transfected with GFP-SKD1 were subjected to immunofluorescence analysis as described in A. (C) HeLa cells transfected with GFP-rab5bQL were subjected to immunofluorescence analyses with anti-SBP1. Upper panel, fluorescence micrographs of GFP-rab5bQL, indicating the enlarged endosomes. Lower panel, Cy3 fluorescence micrograph of SBP1, showing the cytoplasmic localization. Bars: 20 µm.

View larger version (90K): [in a new window]   Fig. 5. Localization of GFP-SBP1 to the E235Q compartments requires C terminus coiled-coil domain. HeLa cells transfected with plasmids encoding GFP-SBP1 deletion mutants and SKD1(E235Q) were subjected to immunofluorescence analysis using anti-SKD1 antibody. Left column (black and white), GFP fluorescence micrographs of the cells transfected with GFP-SBP1 (1-309) (A), C (1-129) (B) and N (198-309) (C). Right three columns, GFP (green), Alexa594 fluorescence (red) and the merged images were obtained from the cells double transfected with SKD1(E235Q) and full-length GFP-SBP1 (1-309) (A), C (1-129) (B) and N (198-309) (C), respectively. Notice, full-length and N SBP1 were redistributed to the E235Q compartments, while C mutant was not. Bar, 20 µm.

View larger version (53K): [in a new window]   Fig. 6. SKD1(E235Q) increases the membrane association of lyst and enlarges lysosomes, but neither SKD1 nor SBP1 can decrease the size of lysosome in CHS fibroblast. (A) The pAb against murine Beige protein can detect human lyst protein on immunoblotting. Total cell lysates prepared from control human fibroblast (GM05655) and fibroblast cells obtained from a CHS patient (GM02075) were subjected to 5% SDS-PAGE and processed for immunoblotting. (B) PNS, cytosol (sup.) and membrane (ppt.) fractions prepared from control and GFP SKD1(E235Q) adenovirus-infected U251 cells were subjected to immunoblotting analysis with anti-lyst antibody. The numbers above each lane represent the volume ratio of all fractions loaded on the SDS gel. Notice, a significant amount of lyst was associated with the membrane in the cells expressing GFP-SKD1(E235Q). (C) Overexpression of GFP SKD1(E235Q) in control fibroblast cells (asterisks in a and b) formed enlarged lysosomes (arrows in a and b), which resembled the phenotype seen in CHS fibroblasts (arrows in c-f). However, neither wild-type GFP SKD1 (asterisks in c and d) nor GFP-SBP1 (asterisks in e and f) can decrease the size of giant-lysosomes in CHS fibroblasts. Bar, 20 µm. (D) Transfection of GFP SKD1(E235Q) in CHS fibroblasts did not inhibit the formation of or redistribution of SBP1 to E235Q compartments (asterisk indicated transfected cell). Scale bar: 20 µm.

View larger version (44K): [in a new window]   Fig. 7. N-terminal coiled-coil domain of mVps2 is required for the formation of E235Q compartments but not for its binding to SKD1. (A) Single and co-transfection of full-length mVps2-myc (a-c), N-mVps2-myc (d-f) and C-mVps2-myc (g-i) with wild type (b, e and h) or SKD1(E235Q) (c, f and i) in HeLa cells. The transfected cells were subjected to immunofluorescence microscopy with anti-myc and anti-SKD1 antibodies. The distribution of single transfected myc-tagged mVps2 variants is indicated in black and white images (a, d and g), while the merged images from the double transfected cells show the localization of myc-tagged mVps2 variants (red) and SKD1 (green). Scale bar: 20 µm. (B) Proportion of cells with E235Q compartments among those double transfected with SKD1(E235Q) and one of the mVps2 variants, tagged with either GFP (green bar) or myc (blue bar). Mean±s.d. values of 3-5 independent experiments are shown. (C) GST pull-down analyses of mVps2-GFP chimeras. Top panel shows immunoblots of cell lysates prepared from HeLa cells transiently expressed full-length mVps2-GFP (lane 1), C-mVps2-GFP (lane 2) and N-mVps2-GFP (lane 3). The cell lysates were incubated with GST-SKD1 (middle panel) or GST alone (bottom panel) adsorbed to glutathione-Sepharose beads. The bound proteins were eluted and immunoblotted with anti-GFP antibody. Asterisks indicated the GFP fusion protein bands of the predicted sizes, 61, 57 and 55 kDa, respectively.