Export out of the endoplasmic reticulum (ER) involves the Sar1 and COPII machinery acting at ER exit sites (ERES). Whether and how cargo proteins are recruited upstream of Sar1 and COPII is unclear. Two models are conceivable, a recruitment model where cargo is actively transported through a transport factor and handed over to the Sar1 and COPII machinery in ERES, and a capture model, where cargo freely diffuses into ERES where it is captured by the Sar1 and COPII machinery. Using the novel secretion inhibitor FLI-06, we show that recruitment of the cargo VSVG to ERES is an active process upstream of Sar1 and COPII. Applying FLI-06 before concentration of VSVG in ERES completely abolishes its recruitment. In contrast, applying FLI-06 after VSVG concentration in ERES does not lead to dispersal of the concentrated VSVG, arguing that it inhibits recruitment to ERES as opposed to capture in ERES. FLI-06 also inhibits export out of the trans-Golgi network (TGN), suggesting that similar mechanisms might orchestrate cargo selection and concentration at the ER and TGN. FLI-06 does not inhibit autophagosome biogenesis and the ER-peroxisomal transport route, suggesting that these rely on different mechanisms.
The authors declare no competing or financial interests.
Y.Y., X.L., K.M., A.K., T.M., P.A., T.F., T.K. performed cell biological experiments and analysis. C.H. and P.H. performed FRAP and STED experiments and analysis, Y.Y. and O.S. performed live-cell imaging and data analysis. R.N. and H.-D.A. synthesized FLI-06. C.K. designed experiments, and C.K. and K.H. discussed the data and wrote the manuscript with the help of all authors. All authors discussed the results and implications at all stages.
This work was supported by a grant from the Deutsche Forschungsgemeinschaft [grant number KA1751/4-1 to C.K.], the Japan Society for the Promotion of Science (to Y.Y.) and Israel Science Foundation [grant number 1533/12 to K.H.].
Supplementary information available online at http://jcs.biologists.org/lookup/doi/10.1242/jcs.186163.supplemental
- Received January 15, 2016.
- Accepted August 25, 2016.
- © 2016. Published by The Company of Biologists Ltd