COPI-coated ER-to-Golgi transport complexes segregate from COPII in close proximity to ER exit sites

Transport of proteins between the endoplasmic reticulum and Golgi apparatus is mediated by two distinct membrane coat complexes, COPI and COPII. Genet ic, biochemical and morphological data have accumulated into a model which suggests a sequential mode of action with COPII mediating the selection of cargo and formation of transport vesicles at the ER membrane for ER-to-Golg i transport and COPI mediating recycling of the transport machinery from po st-ER membranes, To test this transport model directly in vivo, and to study the precise tem poral sequence of COPI and COPII action in ER-to-Golgi transport, we have u sed time lapse microscopy of living cells to visualise simultaneously the d ynamics of COPII and COPI, as well as COPII and GFP tagged secretory marker s in living cells. The majority of COPII labelling appears tightly associat ed with ER membranes that move only within a limited area (less than 2 mu m ) Secretory cargo segregates from these sites and is then transported to th e Golgi apparatus without any apparent association with COPII, COPI-coated transport complexes are seen to form adjacent to the COPII sites on the ER before segregating and moving directionally towards the Golgi apparatus, CO PII is not present on these transport complexes acid remains associated wit h the ER, These data demonstrate for the first time directly in vivo that E R-to-Golgi transport is organised in two steps characterised by a sequentia l mode of action of COPII and COPI.