Cell-Free Analysis

Cell-free systems have been instrumental in the elucidation of the roles of specific cytosolic factors on Golgi apparatus function including events in vesicle formation, transfer and docking (reviewed by Barlowe et al., 1994; Rothman and Warren, 1994). Both permeabilized cells and completely cell-free systems reconstituted from isolated membrane fractions have been used. Completely cell-free systems were applied initially to investigate intra Golgi apparatus trafficking (Fries and Rothman, 1980; Balch et al., 1984a, b; Hammond et al., 1994). Also investigated were transfers between the endoplasmic reticulum and the Golgi apparatus (Nowack et al., 1987; Paulik et al., 1988; Ruohola et al., 1988; Balch et al., 1987; Wuestehube and Schekman, 1992; Barlowe et al., 1994).

As already introduced in Chapters 4 and 8, one of the first contributions of cell-free systems was the identification of a cytosolic factor sensitive to N-ethylmaleimide by Rothman and collaborators (Block et al., 1988; Wilson et al., 1989), referred to as the N-ethylmaleimide-sensitive factor (NSF). A cytosolic protein with homology to the AAA-family of ATPases (Erdmann et al., 1991), NSF consists of a tetramer of four polypeptide chains each with a molecular mass of 76 kDa (Block et al., 1988; Wilson et al., 1989). Additional proteins, e.g., coat proteins, involved in vesicular membrane transfers and targeting also were identified. Clathrin-coated vesicles were shown to mediate transfers among the plasma membrane, endocytic and trans Golgi apparatus compartments (Pearse and Robinson, 1990). The coatomer proteins, COPI i.e. α-, β-, and γ-COPs (Ostermann et al., 1993; Peter et al., 1993; Schekman and Orci, 1996), together with a system of SNAPS and SNAREs (Rothman, 1994; Rothman and Warren, 1994; Subramanian et al., 1996) were subsequently identified and characterized.


Endoplasmic Reticulum Golgi Apparatus Smooth Tubule Acceptor Membrane Transitional Endoplasmic Reticulum 
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© Springer-Verlag Berlin Heidelberg 2009

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