Abstract
The selective export of proteins and lipids from the endoplasmic reticulum (ER) is mediated by the coat protein complex II (COPII) that assembles onto the ER membrane. In higher eukaryotes, COPII proteins assemble at discrete sites on the membrane known as ER exit sites (ERES). Here, we identify Sec16 as the protein that defines ERES in mammalian cells. Sec16 localizes to ERES independent of Sec23/24 and Sec13/31. Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts. Sar1 activity is required to maintain the localization of Sec16 at discrete locations on the ER membrane, probably through preventing its dissociation. Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES.
Highlights
Protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus is dependent on the coat protein complex II (COPII) [1]
In Pichia pastoris [2], and in higher eukaryotes [3,4], assembly occurs at specific sites known as transitional ER or ER exit sites (ERES); the reason for this restricted localization is unknown
There has been some debate as to whether a Sec16 orthologue exists in mammals and the only hint to date has been the identification of a protein of $250 kD that cross-reacts with an antibody directed against the S. cerevisiae Sec16 protein [20]
Summary
Sec has been identified in S. cerevisiae and P. pastoris. The sequences of these genes show highest homology in a central region [18]. These data suggest that, at endogenous levels of expression, Sec requires Sar1GTP for association with ERES; when overexpressed, Venus-Sec can assemble at ERES independent of GTP-loading of Sar. Sec activates Sar with a 10-fold higher turnover rate than the GAP activity of Sec23/24 [33], which suggests that these complexes would have some stability This could result in direct recruitment of Sec (light magenta), or more likely given the discrete localization of Sec to ERES, diffusion of these cargo-Sar1-GTP complexes within the ER membrane, such that they would encounter a Sec assembly and be ‘captured’ (Figure 8C). We hypothesize that assembly of Sec co-ordinates the assembly of the COPII pre-budding complex of Sar, Sec23/24 and thereby provides the spatial coordination for rapid, productive COPII budding events
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