Dynamic of Rab1b and its effectors at ER-Golgi interface

MARTINEZ, H; GARCIA, IA; SAMPIERI, L; ALVAREZ, C

Puerto Natales, CHile

Workshop; EMBO workshop: Current advances in membrane trafficking; 2014

EMBO

Dynamic of Rab1b and its effectors at ER-Golgi interface In eukariotic cells, sorting and concentration of cargo is achieved by the action of COPII (coatomer protein II) complex at specialized ER domains called ER exit sites (ERES). From ERES vesicles and tubules bud and give rise to a compartment called VTCs (vesicle tubular clusters). VTCsundergo maturation by the exchange of COPII for COPI complex and then travel to the cis-Golgi.Rab1b-GTPase is essential for ER-Golgi transport and carries out its function through theinteraction with effectors located at ERES (COPII), VTCs (p115) and Golgi (GM130).Dynamic studies indicate that COPII are relatively immobile and stable structures, while VTCs are mobile and describe trajectories toward and from the Golgi. Interestingly, GFP-Rab1bwt structures exhibit an intermediate behavior between COPII and VTCs. Although individual Rab1b, ERES, and VTCs dynamics have been described, the spatial-temporal connection and comparative dynamics between Rab1b and COPII or p115-labeled structures in living cells have not been explored. In this work, we co-transfected HeLa cells with Cherry-Rab1bwt and YFP-Sec24wt (ERES) or GFP-p115wt (VTCs) to perform time lapse assays using a spinning disk unit. In summary, dynamic analysis indicated that both Rab1b-Sec24 and Rab1b-p115 exhibited transient co-localization showing dynamic subdomains. Furthermore, appearing or vanishing of some Rab1blabelled structures was independent of a COPII or a p115 platform.Moreover, in order to analyze Rab1b-COPII co-localization dynamic in cells unable to recruit COPI, we performed time lapse analysis after brefeldin A (BFA) treatment. Rab1b-COPII co-localization time was significantly increased in the absence of COPI (+BFA). FRAP assays performed on Cherry-Rab1bwt structures showed a rapid recovery (t ½ 20 s) suggesting that COPI recruitment modulate COPII-Rab1b co-localization time without perturbing Rab1b membrane association/dissociation dynamic.This work provides new information about molecular-spatial-temporal mechanisms related to Rab1b participation in ER to Golgi transport.