GBF1 recruitment to membranes: domains and interacting proteins

• Author / Creator

  Chan, Calvin J

• The Golgi complex resides at the center of the cellular trafficking pathway, where it functions in the modification, sorting, and trafficking of over one third of human proteins to their correct cellular destination. Initiation of vesicle formation at the Golgi requires activation of small GTPases of the ADP-ribosylation factor (Arf) family. The Golgi-specific Brefeldin-A resistance factor 1 (GBF1), is the only large guanine nucleotide exchange factor (GEF) that regulates Arf activation at the cis-Golgi and is actively recruited to membranes through a feed forward mechanism upon a reduction in active Arf-GTP.

  Previous studies have demonstrated that GBF1 recruitment requires (a) heat labile and protease sensitive component(s). However, the domains in GBF1 involved in its recruitment, as well as the Golgi-localised protein interactors responsible remain unknown. In this thesis, we first demonstrate using a series of GBF1 deletion mutants that domains HDS1 and HDS2 are essential in the localisation and recruitment of GBF1 to Golgi membranes. To identify GBF1 regulators at the membrane, we further established a method pairing the proximity biotinylation (BioID) based proteomic assay with organelle enrichment to identify Golgi specific GBF1 interacting partners. Unlike past studies on GBF1 interacting proteins, this method allows for the capture of transient, weak, and/or poorly soluble proximal proteins. High-confidence putative interactors were screened via shRNA knockdown and two novel GBF1 interactors were identified and confirmed using coimmunoprecipitation: C10orf76 and ZnT6. Fluorescence and electron microscopy revealed C10orf76 to be a peripheral membrane Golgi-localised protein involved in Golgi maintenance, secretion, and regulation of GBF1 recruitment. C10orf76 exchanges rapidly between free and bound forms, and treatment with the GBF1 inhibitory drug, brefeldin A, increases the residence time of C10orf76 on the membrane. Our phylogenetic analysis suggests that C10orf76 was likely present in the last eukaryotic common ancestor, but subsequently lost from the plant and yeast model systems, Arabidopsis thaliana and Saccharomyces cerevisiae. However, C10orf76 orthologues were identified in other plant and yeast species including Marchantia polymorpha and Schizosaccharomyces pombe. These data suggest that other more evolutionarily conserved GBF1 regulators likely exist. Using fluorescence recovery after photobleaching (FRAP) we demonstrate that another novel regulator, ZnT6, while not essential in recruiting GBF1, plays a role in modulating GBF1 cycling on and off membranes. Knockdown of ZnT6 appears to prevent a fraction of GBF1 from cycling off the membrane. Overexpression reduces the overall fraction of membrane-bound GBF1 and increases the rate of exchange between free and bound forms. We hypothesize that ZnT6 may facilitate the release of GBF1 from the membrane. However, whether this may be due to a direct interaction between ZnT6 and GBF1, or the role of ZnT6 in zinc import into the Golgi lumen remains unclear. Our work has generated new insights into the regulation of GBF1 recruitment to Golgi membranes, and offers a more nuanced picture of the dynamic cellular processes that likely shape GBF1 function in mammalian cells.

• Subjects / Keywords

  • Proteomics
  • Golgi
  • BioID
  • Guanine Nucleotide Exchange Factor
  • Protein Interactions
  • GBF1
  • GTPase

• Graduation date

  Fall 2020

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-z71j-w816

• License

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