COPII–cargo interactions direct protein sorting into ER-derived transport vesicles

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Abstract

Vesicles coated with coat protein complex II (COPII) selectively transport molecules (cargo) and vesicle fusion proteins from the endoplasmic reticulum (ER) to the Golgi complex1,2,3. We have investigated the role of coat proteins in cargo selection and recruitment. We isolated integral membrane and soluble cargo proteins destined for transport from the ER in complexes formed in the presence of Sar1 and Sec23/24, a subset of the COPII components, and GTP or GMP-PNP. Vesicle fusion proteins of the vSNARE family and Emp24, a member of a putative cargo carrier family4, were also found in COPII complexes. The inclusion of amino-acid permease molecules into the complex depended on the presence of Shr3, a protein required for the permease to leave the ER3,5. Resident ER proteins Sec61, BiP (Kar2) and Shr3 were not included in the complexes, indicating that the COPII components bound specifically to vesicle cargo. COPII–cargo complexes and putative cargo adaptor–cargo complexes were also isolated from COPII vesicles. Our results indicate that cargo packaging signals and soluble cargo adaptors are recognized by a recruitment complex comprising Sar1–GTP and Sec23/24.

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Figure 1: Vesicle proteins Hip1, Emp24, Sec22 and gpαF, but not ER resident proteins Sec61, Shr3 and Kar2, are present in complex with Sar1 and Sec23/24.
Figure 2: Vesicle proteins Hip1, Emp24, Sec22 and gpαF, but not ER resident proteins Sec61, Shr3 and Kar2, are present in complex with Sar1 and Sec23/24.
Figure 3: Shr3 is required specifically for incorporation of Hip1 and Gap1 into a complex with Sar1 and Sec23/24.
Figure 4: Shr3 is required specifically for incorporation of Hip1 and Gap1 into a complex with Sar1 and Sec23/24.
Figure 5: Crosslinking of gpαF in a prebudding complex from ER membranes and ER-derived transport vesicles.
Figure 6: Crosslinking of gpαF in a prebudding complex from ER membranes and ER-derived transport vesicles.
Figure 7: Crosslinking of gpαF in a prebudding complex from ER membranes and ER-derived transport vesicles.

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Acknowledgements

We thank S. Bednarek, M. Bowser and L. Liang for performing experiments; P.Ljungdahl, R. Lesch, M. Pilon and A. Corsi for materials; and J. Campbell, T. Doering and other lab members for suggestions. This work was supported by the Howard Hughes Medical Institute (R.S. and M.K.) and the Cancer Research Fund fo the Damon Runyon-Walter Winchell Foundation (M.K.). J.H. was supported by a fellowship from the Deutsche Forschungsgemeinschaft.

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Correspondence to Randy Schekman.

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