RECONSTITUTION OF VESICULATED GOLGI MEMBRANES INTO STACKS OF CISTERNAE - REQUIREMENT OF NSF IN STACK FORMATION

We have developed an in vitro system to study the biochemical events i n the fusion of ilimaquinone (IQ) induced vesiculated Golgi membranes (VGMs) into stacks of cisternae, The Golgi complex in intact normal ra t kidney cells (NRK) is vesiculated by treatment with IQ, The cells ar e washed to remove the drug and then permeabilized by a rapid freeze-t haw procedure. VGMs of 60 nm average diameter assemble into stacks of Golgi cisternae by a process that is temperature dependent, requires A TP and a high speed supernatant from cell extract (cytosol), as reveal ed by immunofluorescence and electron microscopy. The newly assembled stacks are functionally active in vesicular protein transport and cont ain processing enzymes that carry out Golgi specific modifications of glycoproteins. The fusion of VGMs requires NSF, a protein known to pro mote fusion of transport vesicles with the target membrane in the exoc ytic and endocytic pathways, Immunoelectron microscopy using Golgi spe cific anti-mannosidase II antibody reveals that VGMs undergo sequentia l changes in their morphology, whereby they first fuse to form larger vesicles of 200-300-nm average diameter which subsequently extend into tubular elements and finally assemble into stacks of cisternae.