Fig. 1: Schematic illustration showing the role of p125A in endoplasmic reticulum export in mammalian cells. The majority of p125A, Sec31A and Sec13 proteins probably exist in the form of a pre-assembled p125A–Sec31A–Sec13 subcomplex containing two of each of these proteins. Upon recruitment of Sar1 and the Sec23A/Sec24 subcomplex during COPII vesicle budding, the p125A–Sec31A–Sec13 subcomplex is recruited, which may open up the binding sites of p125A for Sec23A. The simultaneous interaction of p125A with both Sec31A and Sec23A on the budding vesicles may facilitate the coordination of these two COPII subcomplexes to mediate vesicle formation.
A team led by Wanjin Hong at A*STAR’s Institute of Molecular and Cell Biology has identified a component of a key molecular complex involved in protein secretion by mammalian cells. “Early work on protein secretion was done mainly in yeast,” says Hong. “Our research is aimed at identifying molecules important for protein secretion in mammalian cells, and at understanding their functional interactions.”
The endoplasmic reticulum (ER) is an extensive membrane network found inside the cell. It encloses an internal space called the lumen from the liquid part of the cell called cytosol. As they are synthesized, proteins targeted for secretion enter the ER lumen where protein folding takes place. They must then be carried within small membrane-enclosed sacs called vesicles to a structure known as the Golgi apparatus, before eventually being secreted from the cell.
In both yeast and mammalian cells, vesicle formation involves a multi-protein complex called coat protein II (COPII), which is assembled at specialized ER exit sites. The last COPII component to be recruited is a subcomplex containing the proteins Sec13 and Sec31 (Fig. 1). “We suspected that the Sec13/Sec31 subcomplex might be linked to molecular mechanisms that regulate vesicle formation at ER exit sites in mammalian cells,” says Hong.
Using the secretion of a viral protein from rat liver cells as a model experimental system, Hong and his co-workers previously found evidence for a Sec31-interacting factor present in the cytosol. They hypothesized that this unidentified factor might be important for regulating export of the viral as well as cellular proteins from the ER lumen.
The team has now identified the factor to be p125A, a protein previously shown to interact with another COPII component known as Sec23. Their new experiments confirmed that p125A interacts directly with Sec31, and that distinct regions of p125A bind to Sec31 and Sec23.
Although p125A is enriched at ER exit sites, further experiments revealed that most of the p125A present in the cytosol is associated with the Sec13/Sec31 subcomplex, suggesting the presence of a pre-assembled complex composed of all three proteins. It was also found that the Golgi apparatus is disrupted and that protein export from the ER is affected in mammalian cells lacking p125A, supporting the view that this protein plays an important role in regulating ER export.
“Future work will test the hypothesis that p125A coordinates the interaction between the Sec13/Sec31 subcomplex and other COPII components such as Sec23 during their recruitment at ER exit sites,” says Hong.
The A*STAR-affiliated researchers contributing to this research are from the Institute of Molecular and Cell Biology.
