Figure 5.

Release of endogenous kinesin from vesicles by Hsc70. V2 vesicles (purified without the use of NEM as described in the text) were resuspended and incubated at a concentration of 1 mg/ml total protein with or without hsc70 for 30 min at 37°C in release buffer (HB plus 75 mM KCl). Hsc70 was used at a concentration of 10 μg/ml for a molar ratio for hsc70:kinesin of 2:1. After incubation, V2 vesicles were recentrifuged at 260,000 × g, and then supernatants and pellets were probed for the presence of kinesin using the H2 antibody on quantitative immunoblots as described above. (a) Purified hsc70 released 50% of the endogenous kinesin remaining on V2 vesicles in a nucleotide-dependent manner. The reaction required Mg-ATP and was blocked by a nonhydrolyzable ATP analogue (AMP-PNP) and EDTA. The amount of kinesin released by hsc70 in this assay was comparable with that released by an antibody against the KLC tandem repeat domain (Stenoien and Brady, 1997). (b) When 2 mM NEM is added to vesicles in vitro at the same time as the hsc70 (time 0), hsc70-mediated release is blocked. The inhibition of hsc70-mediated kinesin release by NEM was eliminated by simultaneous addition of DTT (2 mM). The ability of NEM to inhibit hsc70-mediated release permitted definition of a time course for kinesin release. This could be estimated by adding NEM (2 mM) at the indicated times after the start of incubation, where the start of incubation is the addition of hsc70. When the reaction is allowed to proceed for 5 min before addition of NEM, substantial amounts of kinesin were released. Release of kinesin by hsc70 was essentially complete between 10 and 20 min, so addition of NEM at times >10 min had no effect on the amount of kinesin released by hsc70. (c) The target for NEM modification is a vesicle component, not hsc70 itself. To determine the site of action for NEM, the amount of kinesin remaining bound to vesicles (V) was compared with the amount of kinesin found in the soluble fraction (S) for five different conditions. When vesicles were pretreated with 5 mM NEM, the amount of kinesin released from vesicles by untreated hsc70 (VS pair 2) was similar to that seen without hsc70 (VS pair 1) or with NEM present during the hsc70 incubation (VS pair 3). In contrast, hsc70 pretreated with 5 mM NEM (VS pair 4) exhibited an ability to release kinesin comparable with that of untreated hsc70 (VS pair 5). For pretreatments with NEM, unreacted NEM was neutralized by addition of excess DTT before assay. Error bars represent ±SEM; n = 3 experiments unless otherwise specified.