A Molecular Mechanism for GBF1 Recruitment to cis-Golgi Membranes

• Author / Creator

  Quilty, Douglas

• ADP-ribosylation factors (Arfs) play a central role in the regulation of vesicular trafficking through the Golgi. Arfs are activated on cis-Golgi membranes exclusively by the guanine nucleotide exchange factor (GEF) Golgi-specific BFA resistance factor 1 (GBF1), upon recruitment from cytosol. Membrane association of Arf is essential for its activation; therefore the recruitment of GBF1 to cis-Golgi membranes is a crucial step in Arf activation and Golgi maintenance. Here, we describe a novel mechanism for the regulation of GBF1 recruitment to cis-Golgi membranes. In vivo pharmacological treatments with 2-(4- Fluorobenzoylamino)-benzoic acid methyl ester (Exo1), brefeldin A (BFA), or golgicide A (GCA), as well as transient expression assays including expression of wild-type or mutant Arf and ArfGAP1 constructs, result in selective GBF1 recruitment to cis-Golgi membranes. Specifically, Arf•GDP stimulates further recruitment of GBF1 to Golgi membranes, likely through the activation of a putative GBF1 receptor. The recruitment of GBF1 to Golgi membranes was reconstituted in vitro, which allowed for confirmation of Arf•GDP regulation. Importantly, GBF1 recruited to Golgi membranes by Arf•GDP remains active. Specifically, we demonstrate that GBF1 supports Golgi maintenance, COPI recruitment, and increases Arf activation under Arf•GDP-dependent recruitment conditions. Here we present data suggesting that ArfGAP1 is a critical producer of regulatory Arf•GDP, likely a specialized function at the Golgi. We also determined that Arf•GDP regulates GBF1 recruitment through a mechanism that is dependent on both the N-terminal myristate moiety of Arf•GDP and localisation to the cis-Golgi compartment. Together, these requirements for association of GBF1 with Golgi-membranes suggest regulation of a cis-Golgi membrane bound factor, potentially a protein receptor. Further evidence for the requirement of a Golgi-localised protein was obtained from in vitro experiments in which heat denaturation or protease treatment of Golgi membranes abrogated GBF1 recruitment. We propose that this protein is likely a GBF1 receptor, which would explain the specificity of GBF1 recruitment. Our expectation is that Arf•GDP will positively regulate the GBF1 receptor. A GBF1 truncation library was used to determine that the HDS1 and HDS2 domains of GBF1 are required for association with Golgi membranes and potentially a GBF1 receptor. To identify a putative GBF1 receptor, a far western blot was performed and the results suggest a 32 kDa GBF1 binding partner found on Golgi membranes. In summary, the work described here identified a novel Arf•GDP-stimulated mechanism for GBF1 recruitment and for the first time attributed a role for Arf•GDP in the cell. This mechanism is proposed to support steady-state levels of Arf•GTP at the cis-Golgi during cycles of Arf-dependent trafficking events to maintain Golgi morphology and function. In addition, this work has also yielded novel insight into how BFA potentially acts in vivo. Our increasing understanding of GBF1 recruitment to Golgi membranes provides important insights into how the ERGIC and Golgi compartments are established and maintained.

• Subjects / Keywords

  • GBF1
  • Golgi
  • Trafficking

• Graduation date

  Spring 2017

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R37P8TR8M

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.