Vaults bind directly to microtubules via their caps and not their barrels

Abstract

Vaults are large (13 Mda) ribonucleoprotein particles that are especially abundant in multidrug resistant cancer cells and have been implicated in nucleocytoplasmic drug transport. To understand how these large barrel-shaped complexes are transported through the cytosol, we examined the association of vaults with microtubules both in vitro and in vivo. Within cells, a subpopulation of vaults clearly associates with microtubules, and these vaults remain associated with tubulin dimers/oligomers when microtubules are disassembled by nocodazole treatment. In vitro, a microtubule-pull down assay using highly purified rat vaults and reassembled microtubules reveals that vaults exhibit concentration-dependent binding to microtubules that does not require the carboxyl terminal end of tubulin. Remarkably, negative staining for electron microscopy reveals that vault binding to microtubules is mediated by the vault caps; more than 82% of bound vaults attach to the microtubule lattice with their long axes perpendicular to the long axis of the microtubule. Five to six vault particles were bound per micron of microtubule, with no crosslinking of microtubules observed, suggesting that only one end of the vault can bind microtubules. Taken together, the data support the model of vaults as barrel-shaped containers that transiently interact with microtubules.

Depletion of major vault protein increases doxorubicin sensitivity and nuclear accumulation and disrupts its sequestration in lysosomes

“…As mentioned earlier, there are several observations that would argue that the vault complex is associated with the microtubular network, and perhaps vesicles along it, such as colocalization of MVP and secretory vesicles in neuron-like PC-12 cells (38) and the lysosomal marker CD63 (Lamp-3) in dendritic cells (40). An interesting finding by Eichenmuller et al (37) shows that vaults not only colocalize to microtubules but seem to directly interact with these structures via their structurally more complex cap regions. Two recent reports show the transport of MVP along microtubules (39,50).…”

Vaults and the major vault protein: Novel roles in signal pathway regulation and immunity

“…Both cellular components co-purify from sea urchin cells [30], colocalise to the tips of PC12 neurits [113], and co-immunoprecipitate (with MVP) in non-small cell lung cancer cells [100]. A direct association of a subgroup of vaults with microtubules predominantly via the particle caps was demonstrated with 5 to 6 vaults binding each micron filament [116]. As in this study no vault-mediated crosslinking of microtubules was seen, the authors hypothesised that only one of the vault caps might be able to bind to the filament.…”

“…However, this is to our knowledge the only hypothesis so far published that the two vault halves could be different. Mammalian vaults remained bound to tubulin di-and oligomers even after filament destruction by nocodazole [116], and tubulin was enhanced in the MVP immunoprecipitates after microtubule stabilisation with taxol [100]. However, the intracytoplasmic movement of vaults was only reduced but not completely blocked by nocodazole, suggesting that vault transport, though stimulated, is not entirely dependent on intact microtubules [100].…”

Movement of vault particles visualized by GFP-tagged major vault protein

“…Moreover, sea urchin vaults copurify with microtubules as described by Hamill and Suprenant (1997). In mammalian cells, vaults remain associated with tubulin oligomers even after nocodazol treatment (Eichenmüller et al 2003). Using an in vitro microtubule pull-down assay and negative staining, Eichenmüller et al (2003) have demonstrated that 5-6 vaults bind to 1-μm-long microtubules via their caps.…”

“…Therefore, vault interactions with cytoskeletal elements and molecular motors may be necessary for vault transport over longer distances. An association of vaults with microtubules both in vivo and in vitro has been reported (Hamill and Suprenant 1997;Eichenmüller et al 2003). Furthermore, the colocalization of vaults and microtubules in PC12 and Chinese hamster ovary (CHO) cells has been described ).…”