Forward and retrograde trafficking in mitotic animal cells - ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures

Protein transport arrest occurs between the ER and Golgi stack of mitotic a nimal cells, but the location of this block is unknown. In this report we u se the recycling intermediate compartment protein ERGIC 53/p58 and the plas ma membrane protein CD8 to establish the site of transport arrest, Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII componen t Sec13p, as shown by immunoelectron microscopy, indicating that COPII budd ing structures are the target for mitotic inhibition. The extent of recycli ng of Golgi stack residents was also investigated. In this study we used ol igosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifica tions conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly sy nthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of E R-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi st ack proteins via translocation to the ER.