Golgi apparatus isolation and use in cell-free systems

Transport and internal organization of membranes: vesicles, membrane networks and GTP-binding proteins

“…In these cells proteins can be transferred from one Golgi stack of one parental cell to the Golgi stack of a second cell, supporting that this requires the participation of vesicles (Rothman et al, 1984). This line of reasoning has recently been challenged (Mellman and Simons, 1992;Morré et al, 1993). Moreover, in cell fusion experiments with heterokaryons, transport from the ER to the Golgi is preferentially homologous rather than heterologous, implying that any vesicles involved in this step are not 'free' to diffuse in the cytoplasm (Valtersson et al, 1990).…”

“…As I previously stated, the purification of in vitro vesicles is an argument to support their presence in living cells. This however was critizised by Morré et al (1993) who proposed that in contrast with transition vesicles, clathrin coated vesicles or secretory vesicles, the buds of the intraGolgi apparatus are always associated with the isolated Golgi stacks, even under conditions optimized for interGolgi transfer, and they require harsh extraction methods for their preparation. This point of view was confirmed by experiments in which GTPγS uncoating reaction is accompanied with the formation of vesicles always attached to the cisternae (Weidman et al, 1993).…”

“…The continuity of membrane structures such as the nuclear envelope and the ER, the ER and the dyctiosomes or the annulate lamellae is well known (Watson, 1955;Maul, 1970;Sitia and Meldolesi, 1992). The isolation of the stacks of the Golgi complex and the isolation of the Golgi from the ER and other organelles is becoming controversial (Novikoff and Novikoff, 1977;Broadwell and Cataldo, 1983;Rogalski et al, 1984;Cooper et al, 1990;Mellman and Simons, 1992;Rambourg et al, 1992;Clermont et al, 1993;Morré et al, 1993), and many groups have found tubular organelles associated with endocytic and exocytic pathways (McDowall et al, 1989;Rabinowitz et al, 1992). This data opens the possibility that in some of the intracellular transport pathways, movement is effected in tubular structures rather than by vesicles.…”