COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p.
Formation of COPII-coated vesicles at the endoplasmic reticulum (ER) requires assembly onto the membrane of five cytosolic coat proteins, Sec23p, Sec24p, Sec13p, Sec31p, and Sar1p. A sixth vesicle coat component, Sec16p, is tightly associated with the ER membrane and has been proposed to act as a scaffold for membrane association of the soluble coat proteins. We previously showed that Sec23p binds to the C-terminal region of Sec16p. Here we use two-hybrid and coprecipitation assays to demonstrate that the essential COPII protein Sec24p binds to the central region of Sec16p. In vitro reconstitution of binding with purified recombinant proteins demonstrates that the interaction of Sec24p with the central domain of Sec16p does not depend on the presence of Sec23p. However, Sec23p facilitates binding of Sec24p to Sec16p, and the three proteins can form a ternary complex in vitro. Truncations of Sec24p demonstrate that the N-terminal and C-terminal regions of Sec24p display different binding specificities. The C terminus binds to the central domain of Sec16p, whereas the N terminus of Sec24p binds to both the central domain of Sec16p and to Sec23p. These findings define binding to Sec16p as a new function for Sec24p and support the idea that Sec16p organizes assembly of the COPII coat.
Gimeno
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RE
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Espenshade
P
P
Kaiser
C A
CA
D001665:Binding Sites; D022181:COP-Coated Vesicles; D003001:Cloning, Molecular; D018699:Coated Vesicles / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015003:Yeasts / Q000378:metabolism*
D005656:Fungal Proteins; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C096367:SEC16 protein, S cerevisiae; C079037:SEC23 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins