Spatial-Temporal Study of Rab1b Dynamics and Function at the ER-Golgi Interface

PLoS One. 2016 Aug 8;11(8):e0160838. doi: 10.1371/journal.pone.0160838. eCollection 2016.

Abstract

The GTPase Rab1b is involved in ER to Golgi transport, with multiple Rab1b effectors (located at ERES, VTCs and the Golgi complex) being required for its function. In this study, we performed live-cell dual-expression studies to analyze the dynamics of Rab1b and some effectors located at the ERES-Golgi interface. Rab1b occupied widely distributed mobile punctate and tubular structures, displaying a transient overlaps with its effectors and showing that these overlaps occurred at the same time in spatially distinct steps of ER to Golgi transport. In addition, we assessed Rab1b dynamics during cargo sorting by analyzing the concentration at ERES of a Golgi protein (SialT2-CFP) during Brefeldin A washout (BFA WO). Rab1b was associated to most of the ERES structures, but at different times during BFA WO, and recurrently SialT2-CFP was sorted in the ERES-Rab1b positive structures. Furthermore, we reveal for first time that Rab1b localization time at ERES depended on GBF1, a Rab1b effector that acts as the guanine nucleotide exchange factor of Arf1, and that Rab1b membrane association/dissociation dynamics at ERES was dependent on the GBF1 membrane association and activity, which strongly suggests that GBF1 activity modulates Rab1b membrane cycling dynamic.

MeSH terms

  • Brefeldin A / pharmacology
  • Endoplasmic Reticulum / drug effects
  • Endoplasmic Reticulum / metabolism*
  • Golgi Apparatus / drug effects
  • Golgi Apparatus / metabolism*
  • HeLa Cells
  • Humans
  • Protein Synthesis Inhibitors / pharmacology
  • Protein Transport
  • Vesicular Transport Proteins / metabolism*
  • rab1 GTP-Binding Proteins / metabolism*

Substances

  • Protein Synthesis Inhibitors
  • SEC23A protein, human
  • Vesicular Transport Proteins
  • Brefeldin A
  • Rab1B protein, human
  • rab1 GTP-Binding Proteins

Grants and funding

This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), FONCYT (Préstamo BID 2012. PICT-0043), and Secyt (UNC) to CA as PI. CONICET and INC granted fellowships to HM, LS, and IAG, respectively. CA is member of CONICET. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.