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CHMP1 functions as a member of a newly defined family of vesicle trafficking proteins

Vollum Institute, L474, Oregon Health Sciences University, 3181 S.W. Sam Jackson Park Rd, Portland, OR 97201-3098, USA

View larger version (96K): [in a new window]   Fig. 1. Cytoplasmic CHMP1 colocalizes with endosomes. COS7 cells were double-labeled with antibodies to CHMP1 and the indicated trafficking markers, and visualized by indirect immunofluorescence. Labeled structures are early endosomes, TfR and EEA1; late endosomes, Rab7; lysosomes, LAMP-1; medial and trans-Golgi, mannosidase II (Man II); and trans-Golgi, -adaptin (-adap). Note that CHMP1 and TfR patterns are most highly related.

View larger version (54K): [in a new window]   Fig. 2. CHMP1 is peripherally associated with the membranes of early and late endosomes. (A) HEK293 cytoplasmic extract was centrifuged at 100,000 g to produce soluble (S100) (lane 1) and membrane-associated (P100) (lane 2) fractions. The membrane pellet was sequentially extracted with 750 mM NaCl (lane 3), 0.1 M Na2CO3 (lane 4), and then the residual was completely solubilized (lane 5). Comparable cell-equivalents from each treatment were resolved by SDS-PAGE and analyzed by western blot for CHMP1. (B) Human 293 cytoplasmic extract was layered on, and centrifuged through, a Percoll gradient. The indicated numbered fractions were collected from the top and analyzed by western blot for CHMP1 and the other early (EEA1) and late (Rab7) endosome, and lysosome (LAMP-1) markers. Note that CHMP1 overlaps well with EEA1, but is also detectable in more dense fractions.

View larger version (40K): [in a new window]   Fig. 3. CHMP1 physically interacts with human SKD1/VPS4 in vitro and in vivo. (A) CHMP1 and a control GFP protein were radiolabeled by in vitro translation and incubated with purified, immobilized MBP, MBP-SKD1 or MBP-SKD1(EQ). Bound protein was eluted, resolved by SDS-PAGE and visualized by fluorography. The input lane represents one-tenth the amount of radiolabeled protein used in each binding reaction. Note that only CHMP1 efficiently binds both wild-type and mutant (EQ) SKD1. (B) COS7 cells were transiently transfected with plasmids that produce unfused- or SKD1(EQ) -fused FLAG epitope, plus (lanes 1-4) or minus (lanes 5,6) a CHMP1 expression vector. Cell extracts were immunoprecipitated with anti-FLAG M2 affinity gel (Sigma), and bound material was analyzed by SDS-PAGE, followed by western blot with CHMP1 antibody. Note that SKD1(EQ) can immunoprecipitate endogenous CHMP1 (lane 6). (C) Transfected COS7 cells overexpressing GFP-SKD1(EQ) were analyzed by immunocytochemistry for changes in the endogenous CHMP1 distribution. Transfected (arrowhead) and untransfected (arrow) cells are indicated. Cells were visualized by fluorescence microscopy.

View larger version (65K): [in a new window]   Fig. 4. GFP fusions of CHMP1 and ATPase-defective human SKD1 produce similar effects on endosome structure. COS7 cells were transiently transfected with plasmids encoding the indicated GFP-fusion proteins, fixed, and labeled with antibodies to the indicated proteins or lipid, plus nuclear dye Hoechst 33258. Transfected (arrowheads) and endogenous patterns (arrows) are indicated. (A) Cell were analyzed by phase-contrast and fluorescent microscopy. Note the GFP-positive, phase-contrast visible structures in the cytoplasm that are produced by both CHMP1 and SKD1(EQ) fusions. (B) The indicated antigens were visualized by indirect immunofluorescence microscopy. EEA1 (early endosome antigen 1), a marker for early endosomes (Mu et al., 1995); lysobisphosphatidic acid (LBPA), late endosomes (Kobayashi et al., 1998); and cationic-independent mannose-6-phosphate receptor (M6PR) present on both early and late endosomes.

View larger version (14K): [in a new window]   Fig. 5. CHMP1 is a conserved member of a structurally related, but divergent, family of yeast and mammalian proteins. (A) The six yeast Chm proteins show related size and predicted isoelectric point (pI) values for their N- and C-terminal halves. Yeast CHM open reading frame assignments are CHM1, YKR035w; CHM2, YKL002w; CHM5, YDR486c; and CHM6, YMR077c. (B) Phylogenetic tree for proteins of the yeast Chmp (boxed) and human CHMP families is shown. ClustalX was used to construct the tree (Thompson et al., 1997). D is a measure of sequence divergence. GenBank accession numbers for human proteins are: CHMP1, AF281063; CHMP1.5, AF281064; CHMP2 (BC-2), AF042384; CHMP2.5 (CGI-84), AF151842; CHMP3 (NEDF), AF219226; CHMP4 (HSPC134), AF161483; CHMP5 (CGI-34), AF132968; and CHMP6, AW965590.

View larger version (92K): [in a new window]   Fig. 6. Yeast chm deletions show delayed processing and partial secretion of CPY and produce a Class E vps multi-lamellar structure. (A) Parental yeast (+; BY4741), derivatives generated by deletion of the indicated CHM open reading frames, and the structurally unrelated class E vps sorting mutant, vps4, were pulse-chase labeled as spheroplasts. Intracellular (I) and extracellular (E) fractions were immunoprecipitated with CPY monoclonal antibody (Molecular Probes), resolved by SDS-PAGE, and visualized by fluorography. Proenzyme (pro, 69 kDa) and mature (m, 61 kDa) forms of CPY are indicated. Note that all mutants missort CPY and that the extent of missorting is not increased with a double chm mutant (chm1 snf7). (B-D) Transmission electron micrographs of parental (B) and chm1 mutant cells (C,D). An example of a class E structure (C, arrow) is shown magnified (D). A vacuole in each cell is indicated (v). (E) The frequency (%) of cells with one or more of these multi-lamellar class E vps structures is tabulated for each chm mutant. n, number of cells scored.

View larger version (77K): [in a new window]   Fig. 7. chm deletions in yeast cannot sort a CPS-GFP fusion protein to the vacuole lumen. Parental yeast (+) and the indicated chm mutants were transformed with a GFP-CPS expression vector (2 µm). Each strain was stained with the vital dye FM4-64 (red), which labels the vacuolar membrane under our chase conditions, and was imaged by laser scanning confocal microscopy. Rescue of chm1 is shown with a single copy plasmid containing the ADH promoter fused to the CHM1 open reading frame. Note the lumenal GFP signal in the parent and the CHM1/chm1 strain, but membrane localization in all chm mutants. The strong GFP signal that accumulates adjacent to the vacuole in all chm mutants marks apparent class E compartments.