We microinjected specific antibodies against β-COP to further dissect the role of coatomer in membrane traffic in living mammalian cells. We show here that microinjected antibodies directed against the EAGE-peptide of β-COP block BFA-induced transfer of resident Golgi enzymes to the ER, presumably by inhibiting the fusion of Golgi with ER membranes. Interestingly, relocation of KDEL receptor, a membrane protein cycling between the cis
-Golgi and the IC, is not affected in these cells. Together with our previous results, which show inhibition of anterograde protein transport from the ER/IC to the Golgi complex by the same antibodies (Pepperkok et al., 1993
), these results indicate that COPI is involved in the regulation of both anterograde (IC to Golgi) and retrograde (Golgi to ER) membrane traffic in the early exocytic pathway.
Anti-EAGE stabilizes in vitro binding of coatomer to membranes at least as efficiently as GTPγS, and in vivo, a significant fraction of coatomer remains bound to aggregates of membranes in cells treated with BFA. Out of two dozen antibodies raised against peptides along the sequence of β-COP only two (anti-EAGE, anti-110-12) bound to coatomer when injected into cells and only anti-EAGE interferes with coatomer function (Pepperkok et al., 1993
). The other epitopes must thus be buried inside a protein fold, or are inaccessible within the stable hetero-oligomeric protein complex. Indeed, upon reversible disassembly of coatomer into its monomeric subunits these “hidden” epitopes of β-COP become accessible to the respective peptide antibodies (Lowe and Kreis, 1995
). Thus, the domain around the EAGE-epitope appears to be a major site for heterologous liaisons involved in regulating membrane binding of coatomer. Anti-EAGE may either mask the site where a factor binds β-COP that regulates dissociation of coatomer from its membrane receptor(s), or interfere with a conformational change of β-COP essential for dissociation of coatomer from membranes. While strong evidence suggests that a subcomplex of coatomer composed of α-, β′-, and ε-COP binds to membrane proteins with an ER-retrieval motif (Cosson and Letourneur, 1994
; Lowe and Kreis, 1995
), and that another coatomer subcomplex (containing ζ- and γ-COP) may interact with members of the p24 membrane proteins with a putative “phenylalanine” anterograde transport motif (Fiedler et al., 1996
; Harter et al., 1996
), our data indicate that β-COP must be intimately involved in the regulation of membrane binding of the coatomer complex. Interestingly, binding of the α-, β′-, and ε-COP subcomplex to membranes in vitro is insensitive to GTPγS (Lowe and Kreis, 1995
). It is possible that β-COP, perhaps together with δ-COP with which it interacts directly in the complex (Lowe and Kreis, 1995
), may be involved in conferring ARF-dependent, GTP sensitivity to coatomer–membrane interaction.
In normal cells at steady state, membrane-bound coatomer visualized with antibodies against β-COP is on vesicular structures scattered throughout the cytoplasm or closely attached to, and surrounding, the Golgi complex (Duden et al., 1991
; Kreis et al., 1995
). Interestingly, while about half of the β-COP coated vesicular structures that can be identified immediately upon shifting tsO45-VSV–infected cells to the permissive temperature (after accumulation of ts-O45-G in the IC at 15°C) colocalize with ts-O45-G, the other half appears not to contain viral glycoprotein and may thus be recycling vesicles (Griffiths et al., 1995
). This observation is consistent with the hypothesis that coatomer is involved in both anterograde and retrograde membrane transport. When injected anti-EAGE interfere with transport from the IC to the Golgi complex, cargo (e.g., ts-O45-G) is found in tubular structures containing ERGIC53 and β-COP (Pepperkok et al., 1993
). However, when anti-EAGE interferes with BFA induced relocation of resident Golgi membrane proteins to the ER, virtually no overlap of β-COP with ERGIC-53 and KDEL receptor is observed, yet coatomer closely colocalizes with “Golgiderived cargo.” In both situations no obvious vesicular structures accumulate, but numerous aggregates can be seen with which β-COP is associated (see also Pepperkok et al., 1993
; Oprins et al., 1993
). Since these aggregates are significantly more abundant in injected, BFA-treated cells and contain Golgi resident membrane proteins, but not recycling KDEL receptor, ERGIC53, or ER proteins, they are most likely Golgi derived. These results are thus consistent with genetic data from yeast suggesting that coatomer is involved in regulating membrane transport from the Golgi complex to the ER.
Interestingly, injected anti-EAGE neither leads to accumulation of KDEL receptor in the Golgi complex in normal cells, nor inhibit BFA-dependent relocation of KDEL receptor to the IC. It has been reported that at steady state most of the KDEL receptor is localized to the cis
-Golgi (Lewis and Pelham, 1992
; Tang et al., 1993
). Since the BFA-induced relocation of Golgi resident membrane proteins to the ER/IC aggregates is inhibited by injected antiEAGE, direct retrograde transport of KDEL receptor to the IC and BFA-induced transfer of Golgi resident proteins to the ER must follow independent routes. The simplest explanation for this finding is that the bulk of the KDEL receptor cycles at, or between, the interfaces between the ER (IC) and the Golgi complex (cis
-Golgi network; see also Griffiths et al., 1994
; Tang et al., 1995
), and that in this cycle, recycling is independent of COPI that is recognized by anti-EAGE. Anti-EAGE will only affect relocation to the ER of resident Golgi proteins. This observation in fact further supports a role for COPI in anterograde early secretory membrane traffic. If recycling of an essential factor were inhibited by injected anti-EAGE, then KDEL receptor retrieval should also be affected. It is possible that in normal cells anti-EAGE affects anterograde COPI-dependent membrane traffic more effectively, because it binds to this form of coatomer with higher affinity. This possibility is fully consistent with the hypothesis that coatomer is involved in more than one transport step, and that for each distinct transport step, coatomer has different conformation or composition (i.e., different posttranslational modification of subunits, different subunit isoforms, etc.; see Whitney et al., 1995
; Scheff et al., 1996; Fiedler et al., 1996
; Lowe and Kreis, 1996
We consider it likely that anti-EAGE inhibits the budding of COPI-coated vesicles in vivo. Indeed, the number of COPI-coated vesicles decreases significantly in microinjected cells (Pepperkok et al., 1993
). This is in contrast to the action of GTPγS, which also stabilizes binding of coatomer to membranes and inhibits BFA-induced relocation of resident Golgi proteins to the ER in permeabilized cells (Donaldson et al., 1991
), but does not interfere with the formation of COPI-coated vesicles in vitro (Melançon et al., 1987
). The nature of the accumulating tubulo-vesicular clusters in BFA-treated cells injected with anti-EAGE (many of which remain after wash-out of the drug) is unclear; they probably represent ER/IC membranes that accumulate when early exocytic membrane traffic is inhibited by the injected antibodies and membranes pile up as COPI-coated vesicles cannot bud (see also Pepperkok et al., 1993
). On the other hand, the aggregates (containing resident Golgi transmembrane proteins) that form in the BFA-treated cells injected with anti-EAGE are most likely remnants of Golgi complex derived membranes which cannot fuse with ER membranes as a consequence of the antibody induced stabilization of membrane bound coatomer. It is tempting to speculate that components of the membrane fusion machinery (e.g., v-SNAREs) are present in these aggregates and that they are inactive while covered by coatomer.
Anti-EAGE inhibits reformation of Golgi stacks upon BFA wash-out to only ~50%. This indicates that two mechanisms may lead to Golgi complex reassembly, one COPI dependent and one COPI independent. This is consistent with previous observations that not all Golgi membranes partition into the ER upon treatment of cells with BFA (Oprins et al., 1993
; Orci et al., 1993
; Hendricks et al., 1993
). In addition, two functionally different domains have been predicted within Golgi cisternae based on two different pathways leading to mitotic Golgi disassembly (Misteli and Warren, 1995
), and postmitotic reassembly of the Golgi complex is regulated by two distinct fusion events depending on NSF-SNAPs-p115 and p97 (Rabouille et al., 1995
; Acharya et al., 1995
). Interestingly however, although coatomer is stably bound to membranes of the Golgi complex in cells injected with anti-EAGE, and coatomerdependent budding of vesicles appears inhibited, BFA still leads to the complete disappearance of stacked Golgi cisternae. This result suggests that the BFA induced morphological changes of the Golgi complex cannot be attributed alone to dissociation of COPI from Golgi membranes. Other factors (e.g., ARF), closer in the cascade of events to the action of BFA, may be more directly responsible for this process.
Microinjected anti-EAGE appear to inhibit membrane traffic from the IC to the cis
-Golgi by a different mechanism from that by which they inhibit BFA-induced relocation of Golgi enzymes to the ER. Only ~50% of the normal transport of newly synthesized ts-O45-G to the Golgi is inhibited by the injected antibodies (Pepperkok et al., 1993
), whereas virtually no BFA-induced transfer of Golgi glycosidases to the ER can be measured in cells injected with anti-EAGE. While the precise reason for this major difference remains unclear, several possibilities can be discussed. It could for example be argued that coatomer alone is essential for all retrograde transport, but to some extent redundant (with COPII) for transport in the anterograde direction. It could also be speculated that significantly more binding sites for coatomer reside on membranes of the Golgi complex (consistent with the predominant localization of COPI to the region of the Golgi complex in normal cells) and that as a consequence inhibition of BFA- induced Golgi to ER transport by injected anti-EAGE is more efficient than is transport from the IC to the cis
Golgi. Anti-EAGE may also more efficiently stabilize Golgi membrane bound coatomer due to different conformations of coatomer subcomplexes (Lowe and Kreis, 1995
; Fiedler et al., 1996
) primed to produce retrograde or anterograde directed transport vesicles. Clearly, further experiments will be required to resolve this conundrum.
Given the recent evidence for a role of a coat immunologically related to COPI in the endocytic pathway (Whitney et al., 1995
; Aniento et al., 1996
) and the presence of COPI subunit isoforms generated by differential phosphorylation (Sheff et al., 1996
), it would not be surprising if different forms of coatomer mediated anterograde and retrograde transport between the ER/IC and the Golgi complex. Alternatively, COPI-coated vesicles may provide a “paternoster” continuously recycling immature cargo, its receptors, as well as membrane proteins of the vesicle docking and fusion machinery between the ER/IC and the Golgi complex. One signal for release of cargo into the Golgi complex would then be its dissociation from chaperones which are part of the sequential quality control machinery (Hammond and Helenius, 1995
). Simultaneous visualization of movement of fluorescently modified cargo and coat proteins in living cells may provide further insight into the mechanisms of regulation of early secretory membrane traffic.