Intermediates in the constitutive and regulated secretory pathways released in vitro from semi-intact cells

Grimes and Kelly Secretory Vesicles Formed In Vitro were mechanically permeabilized using the technique of Martin and Walent (1989) . Fig . 1 defines the fractions used in a typical experiment . PC12 cells were washed, resuspended in a cytoplasm-like buffer, and then passed once through a Balch homogenizer (Balch and Rothman, 1985) which permeabilized >95 % of the cells while retaining their original size and shape (data not shown) . Vesicles that leak into the supernatant (Sl) could be separated from the membranes remaining in permeabilized cells, defined as cell ghosts . The membranes of the cell ghosts were analyzed after more vigorous homogenization, followed by a low speed centrifugation to remove nuclei . The postnuclear supernatant (PIM) derived from the cell ghosts should contain membranes that are either firmly attached or too big to leak through the holes in the plasma membrane . The membranes (P2) isolated from the Sl should be enriched in organelles that are not attached and have escaped from the permeabilized cells . We analyzed markers for the Golgi complex and for synaptic vesicles to test these predictions .