Extensive genetic and morphological evidence has implicated COPI in retrograde vesicular traffic from the Golgi apparatus to the ER. Unfortunately, because the processes of anterograde and retrograde transport are intimately linked in a cycle, it has been difficult to assign roles to certain proteins unambiguously to one or the other limb of the cycle. For this reason, we developed a cell-free system that reproduces both events.
Previously, we established a transport assay that measures the vesicular traffic of yeast α-factor precursor from the ER to the Golgi (Baker et al., 1988
). Dean and Pelham (1990)
showed that the yeast retrieval signal, HDEL, appended to the COOH terminus of the α-factor precursor is sufficient to promote the retrieval of intact, glycosylated precursor (gpαF), to the ER in vivo. We combined our cell-free reaction with the use of an HDEL retrieval signal to produce a convenient and reliable tracer of the round-trip reaction. Completion of retrograde transport was monitored by the conversion of a glucosylated form of the gpαF-HDEL, formed in the ER of a strain deficient in glucose trimming of N-linked glycans, to a trimmed species in the ER of a glucosidase proficient strain. Retrieval in vitro was shown to depend on the HDEL signal and on the receptor for this signal, Erd2p.
To focus on those requirements unique to retrograde transport, we incorporated the observations of Barlowe (1997)
, who showed that COPII vesicles dock and fuse with a crude Golgi membrane fraction in the presence of Sec18p, the Lma1p complex, and Uso1p. Other requirements for anterograde transport were supplied by the membrane fraction. Once in the Golgi membrane, [35
S]gpαF-HDEL was retrieved to an acceptor ER fraction in the presence of cytosol. The requirements for cytosol in the retrograde event were satisfied by pure yeast coatomer and myristylated Arf1p. Other obvious requirements for retrieval, such as the Arf1p nucleotide exchange proteins Gea1p and Gea2p (Chardin et al., 1996
; Peyroche et al., 1996
), and possibly an Arf1p GTPase-activating protein, most likely are provided as peripheral membrane components of the Golgi fraction. Retrieval was inhibited by BFA, and this inhibition was relieved by including Arf1p-GTP in the incubation. Thus, the requirement for Arf1p nucleotide exchange is recapitulated in the reaction.
Arf1p has been suggested to promote COPI vesicle budding by activating phospholipase D (PLD), which hydrolyzes PC to create phosphatidic acid, an acidic phospholipid that attracts coatomer to synthetic liposomes (Ktistakis et al., 1996
). However, we saw no evidence of this pathway in our round-trip reaction. Membranes isolated from spo14
, a yeast mutant missing the standard PLD, displayed normal retrieval in vitro (Waksman et al., 1996
). An independent Ca2+
-dependent PLD in yeast (Waksman et al., 1997
) is unlikely to serve as an alternative in retrograde transport because the retrieval reaction sustained by spo14
mutant membranes was unaffected by excess EGTA. Other targets of Arf1p include phosphatidylinositol-specific kinases. PIP2
produced by these kinases may enhance the action of PLD (Martin et al., 1996
). However, wortmannin, an inhibitor of this class of kinases, had no effect on our retrieval reaction.
We used the round-trip reaction to distinguish roles for three v-SNAREs that have been implicated in ER-to-Golgi traffic: Bos1p, Bet1p, and Sec22p. Our results confirm a role for Bos1p in anterograde targeting, and we suggest that Sec22p serves the equivalent role in retrograde targeting. Surprisingly, Bet1p was required in both directions. Bet1p may potentiate SNARE interactions among anterograde and retrograde partners (Stone et al., 1997
) and may replace Sec22p, which is not essential in vivo, on the retrograde limb. In parallel, we have confirmed the roles of Sed5p and Ufe1p as t-SNAREs in anterograde and retrograde traffic, respectively.
Targeting/fusion of retrograde transport vesicles with the ER was blocked by Sec18p antibody. Thus, whereas both directions in the ER-to-Golgi cycle require Sec18p, its homologue, Cdc48p, acts in its place to promote homotypic fusion of ER membranes (Latterich et al., 1995
). Surprisingly, although these two ATPases act to promote distinct fusion reactions involving the ER, both use the same t-SNARE, Ufe1p (Patel et al., 1998
We have shown previously that COPII vesicles package the full set of v-SNAREs required for both directions of targeting in the ER-to-Golgi cycle (Rexach et al., 1994
). Although these vesicles in principle have the complete apparatus of targeting/fusion to both the Golgi and the ER membranes, they do so only to the former (Rexach et al., 1994
). Clearly, some other element, possibly protein or lipid, contributes to the directionality and specificity of this process. Two candidates are Tip20p and Sec20p, proteins that have been shown genetically to interact with Sec22p (Cosson et al., 1997
; Lewis et al., 1997
). Sec20p is a type II integral membrane protein that contains a lumenal HDEL sequence that ensures its recycling to the ER. Tip20p is a peripheral protein that anchors to the membrane in association with Sec20p. Perhaps Sec22p only becomes activated as a v-SNARE when it partners in the Golgi with Sec20p and Tip20p on their way back to the ER. How could this interaction be limited to the Golgi and not occur in the ER from which all three proteins will be recycled into the anterograde path?
Sec22p interacts with Ufe1p, whereas Bet1p does not (Lewis et al., 1997
). Yet they are both required for retrograde transport. Bet1p and Sec22p may form a cis
-SNARE complex in transport vesicles. Upon interaction with Tip20p and Sec20p, this complex may dissociate releasing Sec22p to engage in a trans
-SNARE complex with Ufe1p. Bet1p may be the chaperone for Sec22p in retrograde transport and interact with Ufe1p only in the absence of Sec22p. In the anterograde direction, Bet1p and Bos1p would partner, forming the cis
-SNARE complex and engaging in a trans
-SNARE complex with Sed5p.
flux may provide a crucial distinction between targeting/fusion in the anterograde and retrograde directions. Although COPII vesicle targeting/fusion to the Golgi membrane requires Ca2+
, the retrograde and homotypic fusion events do not (Rexach et al., 1994
; Latterich et al., 1995
). Syntaxin, the synaptic plasma membrane t-SNARE, is associated with and may regulate a Ca2+
channel to provide Ca2+
for fusion of synaptic vesicles (Leveque et al., 1994
; Bezprozvanny et al., 1995
). Likewise, Sec18p-mediated priming and docking of Bos1p and Sed5p may promote Ca2+
flux to activate fusion of COPII vesicles with the cis
-Golgi membrane in yeast. Perhaps this Ca2+
flux acts to reconfigure the Sec22p–Sec20p– Tip20p complex in preparation for the packaging of an active, retrograde v-SNARE into COPI-coated retrieval vesicles. On return to the ER, this complex would be consumed and not reactivated until the proteins reappear at the Golgi membrane. This speculation makes obvious predictions that could be tested by comparing Sec22p complexes found in COPII and COPI vesicles formed in the round-trip reaction described here.