The Secretory Pathway From History to the State of the Art - Fusion of Biological Membranes and Related Problems - page 9

3.2. Transport into and through the Golgi

Transport of material from the ER to the trans -side of the Golgi
involves passage through the intermediate compartment and the various
subcompartments of the Golgi. In fact, vesicular membrane flow was first
reconstituted in a cell free system by using isolated Golgi membranes
(Balch et al ., 1984b). Coated structures were found to bud preferentially
from the rims of Golgi cisternae. It is now well established that intra-Golgi
transport is mediated by the recruitment to the Golgi membrane of soluble
proteins from the cytoplasm, termed COPI. More recently, it was shown
that COPI is also involved in transport from the ERGIC to the cis -side of
the Golgi (reviewed in: Lowe and Kreis, 1998).
Several experimental data support their function in anterograde trans-
port within the early secretory pathway: (i) Biosynthetic cargo such as
albumin and VSV-G protein (Serafini et al ., 1991b), or reporter molecules
carrying a signal for anterograde transport (Nickel et al., 1998) have been
detected in isolated COPI vesicles. Furthermore, a subpopulation of
COPI-coated vesicles has been characterized morphologically and shown
to contain insulin (Orci et al., 1997). (ii) Quantitative immuno-EM has
shown that the majority of -COP can be detected at the cis -side of the

Golgi (Oprins et al ., 1993). This is consistent with extensive co-localization
of COPI with biosynthetic cargo (VSV-G) on the ERGIC (Griffiths et al .,
1995). After a short release from a 15 ° C block, VSV-G is trapped in
COPI vesicles. (iii) Transport of VSV-G to the Golgi can be blocked by
antibodies against a subunit of the COPI coat (Pepperkok et al ., 1993;
Peter et al ., 1993); (iv) COPI can be recruited to pre-Golgi intermediates
where it initiates segregation of VSV-G protein (Aridor et al ., 1995),
and (v) several mutants of S. cerevisiae that are defective in secretion
show defects in one of the COPI subunits (Duden et al ., 1994; Hosobuchi
et al ., 1992).

β

3.2.1. COPI-Coated Vesicles

In vitro generation of COPI-coated vesicles from isolated Golgi mem-
branes has allowed their biochemical characterization (Ostermann et al .,
1993; Serafini et al ., 1991b). Components that make up the coat of COPI
vesicles are the cytosolic proteins ADP-ribosylation factor 1 (ARF1)
(Serafini et al ., 1991a) and coatomer, a hetero-heptameric protein complex
(Waters et al ., 1991) (Table 1).
ARF1, like Sar1p of the COPII coat, belongs to the family of Ras
GTPases. The coatomer complex migrates at 600 kDa and consists of seven
different subunits, called α - (Gerich et al ., 1995), β - (Serafini et al ., 1991b;