Figure 7.

In vitro budding of Tf-HRP containing vesicles from EEs. Tf-HRP was internalized at 15°C in PC12 cells expressing rab4, Q67Lrab4, and S22Nrab4. After 40 min, the cells were washed and homogenized. (A) Donor fractions were prepared from nontransfected PC12 cells and incubated for 30 min with rat brain cytosol and ATP at 0°C (⋄) and 37°C (♦) or in the absence of rat brain cytosol at 37°C (○). Budding reactions were resolved on sucrose gradients and Tf-HRP activity was analyzed colorometrically. Tf-HRP activity in the 20% sucrose peak, represents the budding of vesicles produced from EEs at 37°C. (B) TfHRP vesicle budding-activity for donor membranes prepared from rab4 cells (▴), Q67Lcells (▪), and S22Nrab4 cells (●) at 37°C. TfHRP activity in the gradient fractions is expressed as % of the conjugate internalized at 15°C and is a representative of 5 independent experiments. (C) Comparison of the 20% sucrose peaks formed at 37°C, revealed the same budding efficiency for donor membranes prepared from rab4 cells, Q67Lrab4 cells and control cells. In contrast, the formation of the Tf-containing transport vesicles in S22Nrab4 cells was inhibited twofold. Bars denote the average budding-efficiency of TfHRP containing transport vesicles recovered in the 20% peak fractions (±SEM, n = 5).