Localization of COPI and ERAS relative to ERES. (A) Colocalization of the COPI marker Sec26 with the ERAS marker Tip20. The strain expressed Sec26-GFP together with Tip20-mCherry. Scale bar, 1 µm. (B) Adjacent localizations of the COPI marker Sec26 and the ERES marker Sec13. The strain expressed Sec26-GFP together with Sec13-DsRed. The arrow marks a COPI structure that partly surrounds an ERES. Scale bar, 1 µm. (C) Adjacent localizations of the ERAS marker Tip20 and the ERES marker Sec13. The strain expressed Tip20-GFP together with Sec13-DsRed. The arrow marks an ERAS structure that partly surrounds an ERES. Scale bar, 1 µm. (D) Relative localizations of the COPI marker Sec26, the early Golgi marker Vig4, and the ERES marker Sec13. The strain expressed Sec26-mCherry, GFP-Vig4, and Sec13-HaloTag. Cells were labeled with JF₆₄₆ before three-color imaging. Scale bars, 1 µm for the regular images and 0.5 µm for the magnified insets. (E) Schematic diagram showing the inferred distributions of COPI and COPII vesicles in P. pastoris relative to the ER and early Golgi. On the right is an enlarged and more detailed view of the ER–Golgi interface. According to this proposal, as COPI vesicles bud from the rims of early Golgi cisternae, they engage with the Dsl1 complex, which is bound to ER-localized SNAREs. The COPI vesicles ultimately fuse with the ER. Meanwhile, COPII vesicles bud from ERES, each of which lies beneath an early Golgi cisterna. Thus, by fluorescence microscopy, the Dsl1 complex marks ring-like ERAS that colocalize strongly with COPI and surround ERES.