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First published online 22 February 2005
doi: 10.1242/jcs.01719

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Identification of a novel tubulin-destabilizing protein related to the chaperone cofactor E

¹ Department of Biochemistry, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA
² Department of Biological Sciences, Lehigh University, 111 Research Drive, Bethlehem, PA 18015, USA

View larger version (41K): [in a new window]   Fig. 1. E-like, a protein related to tubulin-folding cofactor E. (A) Amino acid sequence comparison showing 23% identity between human E-like (El) and cofactor E (Cof E). (B) Evolutionary relationship between currently known cofactor E-related sequences; fly, Drosophila melanogaster; celeg, Caenorhabditis elegans; atha, Arabidopsis thaliana; yeast, Saccharomyces cerevisiae; spombe, Schizosaccharomyces pombe; leisch, Leishmania donovani. (C) Schematic of domains identified in cofactor E and E-like. CAP-gly, glycine-rich cytoskeleton-associated protein domain; LRR, leucine-rich repeat sequence; UBL, ubiquitin-like domain. (D) Northern blot showing distribution of E-like expression in various human tissues. Location of size markers is indicated on the left.

View larger version (30K): [in a new window]   Fig. 2. Functional comparison between E-like and cofactor E. (A) E-like cannot substitute for cofactor E in tubulin folding reactions. 35S-labeled, unfolded -tubulin (left panel) or ß-tubulin (right panel) probes generated by expression in E. coli were presented to CCT by sudden dilution from denaturant in the presence of ATP and GTP plus the indicated cofactors (B,C,D) and either cofactor E (E) or increasing concentrations of E-like (El). 1x, 2x and 4x denote the fold molar excess of E-like compared with cofactor E. (B) E-like cannot substitute for cofactor E in tubulin refolding reactions. Tubulin refolding reactions containing various tubulin-specific chaperones were carried out in the presence of [32P]GTP as described (Bartolini et al., 2002). In A and B, reaction products were resolved by non-denaturing gel electrophoresis. Migration positions of various tubulin-containing species are shown. Note the absence of labeled native tubulin heterodimers (tub) in lanes in which E-like is substituted for E. Dß, cofactor D/ß-tubulin; B, cofactor B/-tubulin. (C) E-like does not stimulate the tubulin-GAP activity of cofactors C and D. The tubulin-GAP activity of cofactors C and D was measured as described (Tian et al., 1999) either alone (open circles), in the presence of cofactor E (filled diamonds) or in the presence of E-like (crosses).

View larger version (82K): [in a new window]   Fig. 3. E-like overexpression leads to microtubule depolymerization and Golgi membrane fragmentation. (A) Double-label immunofluorescence of HeLa cells transfected with EGFP alone (a,b), E-like-EGFP (c,d) or E-like-EGFP followed by a brief incubation with nocodazole (e,f). An anti--tubulin antibody recognized by a Texas Red-conjugated secondary antibody was used for the detection of microtubules and tubulin. Arrows in panels c and e highlight MT destruction in cells overexpressing E-like. (B) HeLa cells transfected with E-like-EGFP (El-EGFP) and visualized with a Texas Red-conjugated antibody recognizing an anti-ß-cop antibody to detect Golgi stacks. Golgi membranes in transfected cells are arrowed. (C) E-like is not a microtubule binding protein. 35S-labeled in vitro translated hGEF-H1 (GEF) and E-like (El) were incubated in the presence of taxol-stabilized microtubules and sedimented through a sucrose cushion. Pellet (P) and supernatant (S) fractions containing equal amounts of protein were resolved by SDS-PAGE. Equivalent aliquots of the starting material are shown in the first two lanes. Molecular mass standards are shown on the left. Bar, 10 µm.

View larger version (70K): [in a new window]   Fig. 4. E-like targets tubulin for degradation via the proteosome pathway. (A) 293T cells were transfected with E-like-EGFP (El-EGFP) or EGFP alone (EGFP). Twenty-four hours following transfection, cells were incubated either in the presence (+) or absence (–) of the proteosome inhibitor MG132 (P.I.). Cell lysates were analyzed by SDS-PAGE and by western blotting using a -tubulin antibody for the detection of endogenous tubulin dimers and a ß-actin antibody as a loading control. (B) HeLa cells were transfected with the identical constructs described in A. Arrows indicate tubulin staining in cells overexpressing E-like. Fixed cells were examined by indirect immunofluorescence for the detection of tubulin and microtubules using a Texas Red-conjugated anti--tubulin antibody. Bar, 10 µm.

View larger version (82K): [in a new window]   Fig. 5. E-like disrupts the tubulin heterodimer in vitro. (A) E-like mediated disruption of the tubulin heterodimer in vitro. Purified E-like (El) and cofactor E (E) were incubated with depolymerized bovine brain tubulin (tub). The reaction products were resolved by non-denaturing (N.D.) gel electrophoresis (upper panel) and SDS-PAGE (lower panel). Note that the absence of a band corresponding to purified cofactor E on the native gel is a result of the migration of this protein towards the cathode under these conditions. 0.1x, 0.3x, 1x, 2x and 5x refer to relative molar concentrations of either E-like or cofactor E compared with tubulin. (B) E-like induces aggregation of tubulin in vitro. [35S]tubulin was incubated with the indicated amounts of E-like (El) and the reaction products were analyzed under non-denaturing conditions. (C) E-like prevents MT assembly in vitro. Micrographs (magnification 1300x) show MTs (arrowed) assembled from axoneme fragments 5-10 minutes after warming samples to 37°C. Pre-incubation with 2 µM E-like significantly reduced the assembly of 11 µM tubulin. MTs did not assemble after preincubation with higher concentrations of E-like. The histogram shows the number of MTs assembled per axoneme end for 11 µM tubulin preincubated for 1 hour with the indicated concentrations of E-like. Data shown are the mean±s.d. from 100 axoneme ends per condition.

View larger version (64K): [in a new window]   Fig. 6. siRNA targeting of E-like leads to an increase in the stability of the microtubule cytoskeleton. (A) Depletion of E-like levels upon siRNA transfection. HeLa cells were transfected with either E-like siRNA (El) or a scrambled RNA-oligo duplex (Sc) and harvested at 48, 72 and 96 hours following transfection. Cell lysates were analyzed by immunoblotting using an E-like-specific antibody and a ß-actin antibody as a control for gel loading. (B) Reduced levels of E-like increase microtubule resistance to nocodazole and to Triton X-100 extraction. a-l, HeLa cells were transfected with either scrambled (Sc) or E-like (El) oligoduplexes and treated with nocodazole to completely depolymerize the microtubule cytoskeleton. At the end of the incubation, cells were restored to drug-free medium, fixed at the time points shown (0-15 minutes), and examined by immunofluorescence using anti--tubulin or anti-acetylated tubulin (acet-tub) antibodies. Inset shows an area of a cell at higher magnification to more clearly illustrate a population of intact microtubules. m,n, Control and E-like silenced cells were fixed after brief extraction with Triton X-100 and examined by immunofluorescence using an anti-acetylated tubulin antibody. (C) E-like siRNA does not modify the dynamics of single microtubules in EGFP--tubulin expressing HeLa cells (no siRNA: 16 cells, 59 MTs; with siRNA: 26 cells, 82 MTs). Bar, 10 µm. Vrs, velocity of rapid shortening; Ve, elongation velocity.

View larger version (38K): [in a new window]   Fig. 7. Reorganization of membranes in E-like depleted cells. (A) Clustering of endocellular membranes upon E-like depletion. Control (Sc) and E-like (El) knocked down cells were stained with antibodies against calnexin, giantin or lamp-2 for the detection of the ER, the Golgi complex and lysosomal membranes, respectively. (B) The ER clusters at the MTOC in E-like depleted cells (arrow). E-like silenced cells were immunolabeled with anti-calnexin and anti-acetylated tubulin for the detection of the ER and the MTOC, respectively. Bar, 10 µm.