Possible Roles of Long-chain Fatty Acyl-CoA Esters in the Fusion of Biomembranes - Fusion of Biological Membranes and Related Problems - page 189

formation of coated transport vesicles and buds are greatly reduced
(Pfanner et al., 1989). Ethanol has a similar effect, but is reversed by addi-
tion of palmitoyl-CoA, suggesting for the first time that budding is acyl-
CoA dependent and that an acyl-transfer mechanism is involved in budding
of coated vesicles from Golgi cisternae. The in vitro transport assay was
further refined by removing cytosolic factors from the Golgi membranes by
low salt extraction, and it was shown that coated buds can accumulate in a
GTP-dependent manner when only purified coatomer and N-myristylated
ARF are added (Ostermann et al., 1993). Subsequent addition of palmitoyl-
CoA results in pinching off of coated vesicles, revealing that acyl-CoA is
sufficient for fusion of the adjoining regions of the lipid bilayer within the
coated bud.
Consequently, investigations were conducted to determine whether
LCACoA are required in other steps in the intra-Golgi transport process
(Pfanner et al., 1990). If acyl-CoA esters only are required in the budding
process, then consumption of coated vesicles should be insensitive to addi-
tion of the non-hydrolyzable acyl-CoA analogue. A primed donor was
formed by preincubating donor membranes in the presence of cytosol, ATP
and palmitoyl-CoA. Subsequent addition of acceptor membranes and the
non-hydrolyzable acyl-CoA analogue did not lead to fusion between the
coated vesicles and the acceptor membrane, whereas preincubation of
donor membranes with acceptor membranes completed transport of the
coated vesicles. These observations suggested that acyl-CoA esters are
required in other steps in the transport pathway, downstream of the
budding process (Pfanner et al., 1990). As described above, the role of NSF
appears to be associated with the actual fusion event. Using purified NSF
and NEM-treated membranes (donor and acceptor) preincubated with
cytosol and ATP, it was shown that NSF membrane binding occurs prior to
inhibition of fusion by the non-hydrolyzable acyl-CoA analogue, suggest-
ing that acylation is required for maturation of the junction between the
transport vesicle and the cisternae. A further detailed kinetic analysis of the
acyl-CoA effect identified an NEM-resistant fusion intermediate occurring
immediately prior to fusion which is insensitive to the acyl-CoA analogue
implying that the step which-ultimately leads to membrane fusion, is inde-
pendent of acyl-CoA esters (Pfanner et al., 1990).
The final step in vacuole inheritance in Saccharomyces cerevisiae is
vacuole fusion in daughter cells, allowing yeast to maintain vacuoles as low-
copy organelles. As in heterotypic fusion NSF and -SNAP have also been

shown to be required in homotypic vacuole fusion in yeast (Haas and
Wickner, 1996). Haas and Wickner (1996) observed that myristoyl-, and
palmitoyl-CoA as well as CoASH supported vacuole fusion in vitro, while
short- and very-long chain acyl-CoA esters did not. Consistent with the

α