Membrane Fusion Events during Nuclear Envelope Assembly - Fusion of Biological Membranes and Related Problems - page 290

There is a possibility that another soluble GTPase acting similarly to
ARF inhibits nuclear vesicle fusion in the presence of GTP S. However,

identification of ARF as the most probable soluble component capable of
inhibiting fusion of GTP

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S-treated membranes (Boman et al., 1996), most
likely rules out this hypothesis. Alternatively, another GTPase(s) may be
required for nuclear vesicle fusion. This GTPase may be soluble, as sug-
gested by the cytosolic requirement for extensive nuclear vesicle fusion, and
detectable cytosolic GTPase activity (Wiese et al., manuscript submitted
for publication). The same study predicts the existence of a membrane-
associated GTPase which stimulates nuclear vesicle fusion in the absence
of cytosol. Association of this GTPase with the membrane is salt-resistant,
but whether it is an integral membrane protein or a strongly anchored
peripheral protein is at present unknown. Interestingly, this GTPase is
believed not to be ARF, as salt-treated vesicles lack detectable ARF, yet
still undergo fusion. It will be of interest to investigate how the membrane-
associated GTPase, putative additional soluble GTPase(s) and/or other
soluble components co-operatively and independently function in nuclear
vesicle fusion.

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4. ANALOGIES BETWEEN NUCLEAR VESICLE FUSION
AND FUSION EVENTS IN INTRACELLULAR
MEMBRANE TRAFFICKING

4.1. Inhibition of Nuclear Vesicle Fusion with the Sulphydryl Modifier,

N -Ethylmaleimide

The sulphydryl alkylating agent, N -ethylmaleimide (NEM) has been
known for several years to have adverse effects on NE assembly. Pre-
treatment of nuclear vesicles with millimolar concentrations of NEM, fol-
lowed by quenching of excess NEM with dithiothreitol, does not affect the
ability of vesicles to bind chromatin but potently inhibits fusion (Collas and
Poccia, 1996b; Macaulay and Forbes, 1996; Newport and Dunphy, 1992;
Vigers and Lohka, 1991). As a result, formation of a double nuclear mem-
brane and assembly of NPCs are prevented (Macaulay and Forbes, 1996).
NEM does not affect post-fusion events in the NE assembly process such
as vesicle flattening or the assembly of NPCs once the membranes have
fused (Macaulay and Forbes, 1996). The ability of the sea urchin fusigenic
vesicle fraction MV1 to bind LSs is also sensitive to NEM treatment of the
vesicles (Collas and Poccia, 1996b). Treatment of LS-bound MV1 vesicles
with NEM also affects LS-MV1 fusion (P.C., unpublished data). The sensi-