Temperature- and acceptor-specificity of cell-free vesicular transfer from transitional endoplasmic reticulum to the cis Golgi apparatus

Abstract

The temperature dependence and specificity of transfer of membrane constituents from donor transitional endoplasmic reticulum to the cis Golgi apparatus were investigated using a cell-free system from rat liver. The radiolabelled transitional endoplasmic reticulum donors were prepared from slices of rat liver prelabelled with [14C]leucine. The acceptor Golgi apparatus elements were unlabelled and immobilized on nitrocellulose. When Golgi apparatus stacks were separated by preparative free-flow electrophoresis into subfractions enriched in cisternae derived from the cis, medial and trans portions of the stack respectively, efficient specific transfer was observed only to cis elements. Trans elements were devoid of specific acceptor capacity. Similarly, when transfer was determined as a function of temperature, a transition was observed in transfer activity between 12 degrees C and 18 degrees C similar to that seen in vivo for formation of the so-called 16 degrees C cis Golgi-located membrane compartment. Transfer at temperatures below 16 degrees C and transfer to trans Golgi apparatus compartments at temperatures either above or below 16 degrees C was similar and unspecific. The unspecific transfer at low temperature was pH independent, whereas specific transfer was greatest at the physiological pH of 7, and was reduced to 10% and 18% of that occurring at pH 8 and pH 5.5 respectively. These findings show that the cell-free system derived from rat liver exhibits a high degree of fidelity to transfer in vivo, an efficiency approaching that observed in vivo, and a nearly absolute acceptor specificity for cis Golgi apparatus. The acceptor-, temperature- and pH-specificity of the cell-free transfer, as well as the saturation kinetics exhibited with respect to acceptor Golgi apparatus, support the concept of transition-vesicle-specific docking sites of finite number associated with cis Golgi apparatus cisternae.

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

“…Thus, specific transfers of 2.5-5% of the starting radioactivity represented a practical upper limit of transfer without first isolating and concentrating the transition vesicles. The percentages of transfer observed in the cell-free system are comparable to those estimated in vivo or with liver slices (Dunkle et al 1992). In vivo, endoplasmic reticulum membranes of rat liver reached a specific radioactivity of about 70 000 dpm/mg of protein over 30 min.…”

“…Trans elements were devoid of specific acceptor activity. Additionally, transfer between transitional endoplasmic reticulum and cis Golgi apparatus exhibited saturation kinetics with respect to acceptor membrane suggesting a limited number of fusion sites (Dunkle et al 1992). Saturation kinetics with respect to acceptor Golgi apparatus (Dunkle et al 1992) support the suggestion that the in vitro-generated vesicles utilize transition vesicle-specific docking sites of finite number associated with the cis Golgi apparatus cisternae (Rothman and Warren 1994).…”

“…Additionally, transfer between transitional endoplasmic reticulum and cis Golgi apparatus exhibited saturation kinetics with respect to acceptor membrane suggesting a limited number of fusion sites (Dunkle et al 1992). Saturation kinetics with respect to acceptor Golgi apparatus (Dunkle et al 1992) support the suggestion that the in vitro-generated vesicles utilize transition vesicle-specific docking sites of finite number associated with the cis Golgi apparatus cisternae (Rothman and Warren 1994). Palade (1983) first suggested that vesicle fusion was a receptor-mediated process which predicted that fusion of donor vesicles with acceptor membranes would exhibit saturation kinetics.…”

“…The liver system is donor and acceptor specific (Nowack et al 1987, Dunkle et al 1992. When Golgi apparatus stacks were separated by preparative free-flow electrophoresis into subfractions enriched in cisternae derived from cis, medial, and trans portions of the stack, efficient transfer of radioactive membrane proteins from radiolabeled transitional endoplasmic membranes was observed only with the cis elements (Dunkle et al 1992).…”

Membrane Flow Revisited

“…All three players participating in endoplasmic reticulum-to-Golgi apparatus transfer-the transitional endoplasmic reticulum, transition vesicles, and eis-Golgi apparatus cisternaehave been isolated as purified, functional fractions from rat liver. Transfer and processing of membrane proteins and Iipids have been reconstituted in cell-free systems using the purified components (Dunkle et al 1992, Moreau and Morre 1991, Paulik et al 1988: Endoplasmic reticulumto-Golgi apparatus transfer in these cell-free systems exhibited a remarkable fidelity to that in living cells in terms of specificity, ATP requirement, pH optimum, and temperature dependence, as well as sensitivity to sulfhydryl reagents, involvement of monomeric GTP-binding proteins, and efficiency of transfer. Compelling evidence for coated vesicular intermediates between the endoplasmic reticulum and the Golgi apparatus has emerged from other studies as well.…”