Figure 6.

The p80 homology region of TEP1 contains the vault interaction domain. (A) TEP1 truncations were co-expressed with the MVP in Sf9 insect cells using a baculovirus expression vector. (B) MVP forms vaults when expressed in insect cells, which are found entirely in the high-speed pellet (P100) of cell lysates and can be purified to near-homogeneity. Vault-interacting proteins will co-purify with vaults. Vaults were further enriched, and as a final step, samples were loaded onto a 20–60% sucrose gradient and centrifuged for 16 h. Sucrose fractions were pelleted, resuspended in 20 mM MES buffer, pH 6.5 and analyzed by western blot. Vaults fractionate largely in the 45% sucrose layer (upper panel). In the absence (−) of MVP-baculovirus co-infection, TEP1 truncations fractionate in either the high-speed supernatant (S100), P100, or both, and are lost in the purification scheme before the sucrose gradient step (1, 2, and 3, upper panels). When MVP is present (+), TEP1 truncations able to interact with vaults are found largely in the 45% sucrose layer (column 1, 2, and 3, lower panels).