Sulfhydryl Involvement in Fusion Mechanisms - Fusion of Biological Membranes and Related Problems - page 495

incubation of the membranes with N -ethylmaleimide. By a number of cri-
teria the N -ethylmaleimide-sensitive component was demonstrated to be
distinct from NSF (Sokoloff et al., 1995). Subjecting the untreated mem-
branes to a procedure known to extract NSF from membranes did not
affect microsome fusion, whereas addition of cytosol that had not
been treated with N -ethylmaleimide did not restore fusion between N-

ethylmaleimide-treated microsomes. NSF was not detected immunologi-

cally in microsomes that retained fusion capacity.
Treatment of the membranes with 3-(2-pyridyldithio)propionate
coupled to bovine serum albumin (BSA-PDP) inhibited microsome fusion
in a reaction that was reversible by subsequent addition of dithiothreitol
(Sokoloff et al., 1995).The BSA-PDP appeared to react with the same thiols
as the N -ethylmaleimide, because it prevented the irreversible inhibition of
fusion by N -ethylmaleimide. These data suggested that the functionally sig-
nificant thiols are highly accessible on the membranes. Inhibition of fusion
could also be achieved through treatments with sodium periodate in a reac-
tion that was also reversible by subsequent addition of dithiothreitol. Incu-
bation of the membranes with 1 mM Mg 2+ -GTP did not protect the fusion
activity of the membranes from inhibition, thereby indicating that the reac-
tive thiol was unlikely to be in a GTP-binding site (Sokoloff et al., 1995).

2.2.7. Endocytosis

N -ethylmaleimide-sensitive factors that differ from NSF have been
reported to participate in vesicular transport and membrane fusion during
endocytosis. In a cell-free system that examines the transport of mannose
6-phosphate receptors from late endosomes to the trans-Golgi network, it
has been demonstrated that transport was 80% inhibited when mixtures
of cell extracts and Golgi membranes were treated with 0.2 mM N -
ethylmaleimide (Goda and Pfeffer, 1991). Transport was largely restored
when untreated, but not N -ethylmaleimide-treated, cytosol was added to
the mixture. Glycerol-gradient sedimentation analysis of the active compo-
nent of the cytosol, dubbed ETF-1, indicated that it had a molecular mass
of 50-100 kDa, whereas NSF is a hexamer of 76-kDa subunits (Fleming et
al., 1998). Furthermore, the ETF-1 activity was still present in cytosol
depleted of NSF by passage over columns containing bound anti-NSF anti-
bodies. In addition, levels of N -ethylmaleimide that inhibited endosome

→
trans-Golgi network transport did not inhibit NSF-dependent intra-Golgi
transport indicating that NSF is not the target of N -ethylmaleimide in the
endosome trans-Golgi network transport system (Goda and Pfeffer,

1991). ETF-1 activity was found to be required for an early stage in trans-
port but has not been further characterized.

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