Cell extracts and microtubule co-sedimentation assay Extracts from HeLa cells infected for 24 h or uninfected controls maintained in the presence of 0.1 mg/ml rifampicin were prepared as described previously (Ro Èttger et al., 1999) . The extract was clari®ed by centrifugation at 150 000 g for 20 min at 4°C and cytochalasin D added to a ®nal concentration of 1 mg/ml to depolymerize actin ®laments. Endogenous tubulin in the extract was polymerized in a two-step procedure. First, the extract supernatant was supplemented with protease inhibitors, 2 mM MgGTP and 5 mM taxol and incubated for 5 min at room temperature; subsequently, an additional 15 mM taxol was added to the mix and the reaction incubated at 33°C for 30 min. For controls, no taxol was added at any stage and microtubule polymerization was inhibited either by the addition of nocodazole to a ®nal concentration of 40 mM or by maintaining the extract at 4°C throughout the experiment. Following microtubule assembly, each 400 mg extract reaction was diluted 5-fold in BRB80 buffer (containing protease inhibitors and 20 mM taxol) and centrifuged through a 10% sucrose cushion containing protease inhibitors and 20 mM taxol at 165 000 g for 20 min at 25°C. The microtubule pellet was solubilized in SDS±PAGE sample buffer and analysed by SDS± PAGE.