TGN recruitment of TRAPPII reveals TRAPP maturation as unlikely to drive RAB1-to-RAB11 transition.

Transport protein particle (TRAPP) complexes regulate membrane traffic. TRAPPII and TRAPPIII share a core hetero-heptamer, also denoted TRAPPI. In fungi TRAPPIII and TRAPPII mediate GDP exchange on RAB1 and RAB11, respectively, regulating traffic across the Golgi, with TRAPPIII also activating RAB1 in autophagosomes. Our finding that TRAPPII can be assembled by addition of a TRAPPII-specific subcomplex onto core TRAPP prompted us to investigate the possibility that TRAPPI and/or TRAPPIII already residing in the Golgi matures into TRAPPII to determine a RAB1-to-RAB11 conversion as Golgi cisternae progress from early Golgi to TGN identity. By time-resolved microscopy, we determine that the TRAPPII reporter Trs120 (the homolog of metazoan TRAPPC9) is recruited to existing trans-Golgi network (TGN) cisternae slightly before RAB11 arrives, and resides for ∼45 s on them before cisternae tear off into RAB11 secretory carriers. Notably, the core TRAPP reporter Bet3 (the homolog of metazoan TRAPPC3) was not detectable in early Golgi cisternae, being instead recruited to TGN cisternae simultaneously with Trs120, indicating recruitment of TRAPPII to the Golgi and arguing strongly against the TRAPP maturation model.