Sulfhydryl Involvement in Fusion Mechanisms - Fusion of Biological Membranes and Related Problems - page 489

comparable effectiveness for reduction of an intermolecular disulfide.
However, a dithiol is kinetically superior as a reductant for intramolecular
cystine bridges when compared to a monothiol (Gilbert, 1990). This fact
may underlie the very different in vivo effects of dithiothreitol treatments
versus 2-mercaptoethanol treatments upon the secretion from mammalian
cells of proteins containing cysteine residues and/or cystine bridges (Valetti
and Sitia, 1994).

2. PROTEIN THIOLS IN CELLULAR MEMBRANE FUSION

2.1. Identified Thiol-Reagent-Modified Proteins

2.1.1. N -ethylmaleimide-Sensitive Factor-NSF

The most prominent protein bearing a thiol whose modification
interferes with cellular membrane-fusion processes is the N-
ethylmaleimide-sensitive factor (NSF). A complete treatment of the roles
of NSF in secretory vesicular transport and its interactions with soluble NSF
attachment proteins (SNAP S ) and SNAP receptors (SNAREs) is beyond
the scope of this review. Numerous recent reviews of the function of these
proteins are available (Clague, 1998; Hay and Scheller, 1997; Nichols and
Pelham, 1998; Robinson and Martin, 1998; Woodman, 1997). However, con-
sideration of a number of points concerning the effects of N -ethylmaleimide
treatment upon its function will be instructive. It is also important to be
familiar with some of the characteristics of NSF, because in publications
that will be discussed below the properties of other membrane-fusion
factors are explicitly contrasted with those of NSF.
Biosynthetic transport between Golgi cisternae in cell-free systems is
inhibited by treatment with 1 mM N -ethylmaleimide and restored by addi-
tion of untreated NSF, which appears to be the inactivation target in the
membranes. NSF is a hexameric ATPase that plays a central role in many
vesicular transport pathways including endoplasmic reticulum-Golgi appa-
ratus transport, transport between Golgi complex cisternae, endosomal
vesicle fusion, and transport from the Golgi to specialized membranes in
polarized cells (Woodman, 1997). The membrane-fusion-promotion and
ATPase activities of purified NSF are inhibited by treatment with 2 mM N -
ethylmaleimide (Block et al., 1988; Tagaya et al., 1993). NSF forms an ATP-
dependent complex with soluble NSF attachment proteins (SNAPs) that in
turn interact with SNAP receptors (SNAREs) and, it is believed, thereby
induce conformational changes that affect SNARE function (Woodman,
1997). It has been recently suggested that NSF may also act as a molecular