Effect of caffeine and reduced temperature (20 degrees C) on the organization of the pre-Golgi and the Golgi stack membranes

Reduced temperature has been widely used to dissect transport steps in the exocytotic transport pathway (for review see Saraste and Kuismanen, 1992) . Specifically, incubation at 15~ blocks the transport at the pre-Golgi level (Saraste and Kuismanen, 1984) and has led to the characterization of p58, a resident protein in the pre-and cis-Golgi compartments (Saraste et al., 1987) . The depletion of cellular ATP has been shown to inhibit distinct steps in vesicular transport (Balch et al., 1986; Tartakoff, 1986) . The important role of calcium in vesicular traffic between the ER and the Golgi compartment has become evident from in vitro studies (Beckers and Balch, 1989; Sambrook, 1990) . Also, the perturbation of intraceUular Ca2+-levels blocks the movement of secretory proteins out from the rough ER (Ix)dish and Kong, 1990 ). In addition, lowered extraceUular pH has been reported to arrest the transport of influenza virus hemagglutinin at the pre-Golgi level (Matlin et al., 1988) . The central role of GTP in the vesicular transport has become evident from in vitro studies where the nonhydrolyz-able GTP-analogue, GTP-3,-S, has been used to inhibit ERto-Golgi transport both in mammalian and yeast ceils (Beckers and Balch, 1989; Ruohola et al., 1988) . Recent localization of the small GTP-binding proteins rablb and rab2 to the membranes responsible for the ER-to-Golgi transport has further emphasized the role of GTP as a regulator of ER-to-Golgi membrane traffic (Chavrier et al., 1990; Plutner et al., 1991) . Interestingly, there is also evidence for the participation of trimeric GTP-binding proteins in the regulation of membrane traffic (Stow et al., 1991; Barr et al., 1991) , thus revealing a link between signal transduction and the processes in the intracellular membrane traffic (for review see Balch, 1992; Barr et al., 1992) . Okadaic acid, a phosphatase inhibitor, has been shown to inhibit ER-to-Golgi transport (Davison et al., 1992) and result in the fragmentation and dispersal of the Golgi apparatus (Lucocq et al., 1991) , further linking mechanisms of signal transduction and the endomembrane traffic.