Vaults bind directly to microtubules via their caps and not their barrels.
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.
Department of Molecular Biosciences, University of Kansas, Lawrence, KS 66045, USA.
Bernd
B
Eichenmüller
Nancy
N
Kedersha
Elena
E
Solovyeva
Patrick
P
Everley
Jennifer
J
Lang
Richard H
RH
Himes
Kathy A
KA
Suprenant
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000378:metabolism; D002458:Cell Fractionation; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D015739:Nocodazole / Q000378:metabolism; D017239:Paclitaxel / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport / Q000502:physiology*; D051381:Rats; D020860:Subtilisin / Q000378:metabolism; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D020394:Vault Ribonucleoprotein Particles / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D000099187:Major Vault Protein
D000972:Antineoplastic Agents, Phytogenic; D015739:Nocodazole; D017239:Paclitaxel; D020860:Subtilisin; D014404:Tubulin; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein