The identification and characterisation of arfophilin-2

Hickson, Gilles Reisan Xavier (2001) The identification and characterisation of arfophilin-2. PhD thesis, University of Glasgow.

Abstract

ADP-ribosylation factors (Arfs) are ubiquitously expressed small molecular weight GTPases whose known functions in mammalian cells are as key regulators of vesicle biogenesis and modulators of the actin cytoskeleton in the Golgi and plasma membrane systems. Previous studies have paid particular attention to the class I Arfl and the class III Arf6, while there is virtually no information on the cellular functions of the class II Arf isoforms, Arf4 and Arf5. While the different Arf isoforms have the same activities in a number of in vitro and in vivo assays, several observations point to specific functions for the class II members. These include the identification of an Arf5-specific guanine nucleotide exchange factor, GBFl, and the identification of Arfophilin, a putative effector protein of unknown function that shows specificity for class II and III isoforms. Here a yeast two-hybrid screen has been performed to identify novel GTP-dependent Arf5 binding proteins. This yielded two previously identified Arf-interacting proteins, Arfaptin 2/PORl and Arfophilin, but also two novel ones, HCR and an Arfophilin homologue that we have named Arfophilin-2. In the yeast two-hybrid system, Arfophilin-2, like Arfophilin-1, appeared to specifically interact with the GTP-bound conformations of class II and III Arfs and did not interact with class I isoforms. HCR displayed unique specificity for Arf5, but was not analysed further. A full-length Arfophilin-2 open reading frame (ORF) was cloned from testis cDNAs, although there is evidence of 5' alternative splicing. Northern blotting revealed that Arfophilin-2 mRNA is highly expressed in testis but also at low levels in many other tissues, while Arfophilin-1 is predominantly expressed in testis, kidney and skeletal muscle. Arfophilin-2-specific polyclonal antibodies were raised against the C-terminal domain and used to analyse the native protein. They recognised a single band with an apparent molecular weight of 83 kDa in HeLa and HEK 293 cells, as well as in a human testis homogenate. Moreover, these bands had the same electrophoretic mobility as the protein product produced from the cloned ORF. Using immunofluorescence microscopy, endogenous Arfophilin-2 was found to reside primarily in a brefeldin A-sensitive, cation- independent mannose-6-phosphate receptor-positive, trans-Golgi network (TGN) compartment. Some staining was also observed at the centrosome, where it partially colocalised with alpha-tubulin, and at the plasma membrane in structures resembling focal contacts or actin-rich protrusions. Overexpression of green fluorescent protein (GFP) fused to the C-terminal Arf-binding domain of Arfophilin 2, or indeed to full-length Arfophilin 2, resulted in a tight pericentrosomal localisation in HeLa, HEK 293 and CHO cells. Strikingly, transferrin receptors (TfRs) and Rab 11, a marker for the perinuclear recycling endosomes, were also redistributed to the same location while early endosomal and Golgi/TGN markers were unaffected. Trafficking of TfRs through the compartment did not seem to be compromised, as evidenced by pulse-chase experiments using fluorescent transferrin in transiently transfected HeLa cells. The pericentrosomal localisation of the GFP-Arfophilin 2/TfR/Rab ll compartment was sensitive to microtubule disruption using nocodazole, which caused the compartment to disperse into punctate structures throughout the cytoplasm. Conversely, Brefeldin A treatment was without discernible effect. Amino acid sequence analysis revealed that the Arfophilins are homologous to Nuclear Fallout (Nuf) in their C-termini. Nuf is a cell-cycle regulated protein that plays a critical role in Drosophila melanogaster cellularisation where it is believed to facilitate the loading of vesicles onto microtubules at the centrosome for subsequent transport to the periphery. GFP-Nuf constructs were engineered and expressed in HeLa cells to see whether a similar phenotype would arise. Strikingly, the same clustering of TfRs and Rab 11 was observed, again without noticeably compromising the ability of fluorescent transferrin to traffic through the compartment. These data suggest that Nuf and Arfophilins are functionally homologous. In summary, this work has uncovered a novel putative Arf effector protein and has provided the first functional analysis of the mammalian Arfophilins. The results suggest they may be important in regulating the spatial distribution of the endosomal recycling compartment, and by analogy with the Nuf protein, they may be involved in regulating membrane trafficking during cytokinesis.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Keywords: Biochemistry
• Subjects: Q Science > QH Natural history > QH345 Biochemistry
• Colleges/Schools: College of Medical Veterinary and Life Sciences
• Supervisor's Name: Gould, Professor Gwyn
• Date of Award: 2001
• Depositing User: Enlighten Team
• Unique ID: glathesis:2001-72684
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 13 Jul 2022 14:17
• Thesis DOI: 10.5525/gla.thesis.72684
• URI: https://theses.gla.ac.uk/id/eprint/72684