Abstract
COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sar1 GTPases, respectively. We show that COPI and COPII coat complexes are recruited separately and independently to ER (COPII), pre-Golgi (COPI, COPII), and Golgi (COPI) membranes of mammalian cells. To address their individual roles in ER to Golgi transport, we used stage specific in vitro transport assays to synchronize movement of cargo to and from pre-Golgi intermediates, and GDP- and GTP-restricted forms of Sar1 and ARF1 proteins to control coat recruitment. We find that COPII is solely responsible for export from the ER, is lost rapidly following vesicle budding and mediates a vesicular step required for the build-up of pre-Golgi intermediates composed of clusters of vesicles and small tubular elements. COPI is recruited onto pre-Golgi intermediates where it initiates segregation of the anterograde transported protein vesicular stomatitis virus glycoprotein (VSV-G) from the retrograde transported protein p58, a protein which actively recycles between the ER and pre-Golgi intermediates. We propose that sequential coupling between COPII and COPI coats is essential to coordinate and direct bi-directional vesicular traffic between the ER and pre-Golgi intermediates involved in transport of protein to the Golgi complex.
Highlights
COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sarl GTPases, respectively
We used mutants of ARF1 and Sarl proteins which are restricted to GDP- or GTP-bound forms to selectively control COPI and COPII assembly and disassembly. We find that both coats are required to transport the same cargo molecule, in this case vesicular stomatitis virus glycoprotein (VSV-G), from the ER to the Golgi stack
To study the role of COPI and COPII coats in ER to Golgi transport, we first analyzed the ability of crude microsoreal membranes to bind coat complexes after incubation in vitro and centrifugation to separate membrane-bound and cytosolic forms of coat complex components
Summary
COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sarl GTPases, respectively. Studies in vitro (Kuge et al, 1994; Peter et al, 1993, 1994) and in vivo (Guo et aL, 1994; Hobbie et al, 1994; Pepperkok et al, 1993) in mammalian cells, and biochemical and genetic analyses in yeast (for review see Pryer et al, 1992), have shown that both coats are essential for the anterograde transport of protein from the ER to the Golgi stack Their individual specific roles in mediating the movement of cargo through early steps of the secretory pathway remains to be determined. CGN, cis-Golgi network; GAP, guanine nucleotide activating protein; GTP~S, guanosine 5'-O-(3-thiotriphosphate); myr, myristoylated; VSV-G, vesicular stomatitis virus glycoprotein; VTC, vesicular tubular cluster
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