1. Class II Arfs require a brefeldin-A-sensitive factor for Golgi association
- Author
-
John F. Presley and Selma Y. Dejgaard
- Subjects
0301 basic medicine ,GTP' ,Biophysics ,Golgi Apparatus ,GTPase ,Biochemistry ,Clathrin ,03 medical and health sciences ,symbols.namesake ,chemistry.chemical_compound ,0302 clinical medicine ,Vesicle uncoating ,Guanine Nucleotide Exchange Factors ,Humans ,HA-tag ,Molecular Biology ,Brefeldin A ,biology ,ADP-Ribosylation Factors ,Chemistry ,Cell Biology ,COPI ,Golgi apparatus ,Cell biology ,030104 developmental biology ,030220 oncology & carcinogenesis ,biology.protein ,symbols ,ADP-Ribosylation Factor 1 ,HeLa Cells - Abstract
Arf proteins are small Ras-family GTPases which recruit clathrin and COPI coats to Golgi membranes and regulate components of the membrane trafficking machinery. It is believed membrane association and activity of Arfs is coupled to GTP binding, with GTP hydrolysis required for vesicle uncoating. In humans, four Arf proteins (Arf1, Arf3, Arf4 and Arf5) are Golgi-associated. Conflicting reports have suggested that HA-GFP-tagged Class II ARFs (Arf4 and Arf5) are recruited to membrane independently of the brefeldin A sensitive exchange factor GBF1, suggesting regulation fundamentally different from the Class I Arfs (Arf1, Arf3), or alternately that the GTPase cycle of GFP-tagged Class II Arfs is similar to other Arfs. We show that these results depend on the fluorescent tag, with Arf4-HA-GFP tag resistant to brefeldin, but Arf4-GFP acting similarly to Arf1-GFP in brefeldin-sensitivity and photobleach assays. Arf4-HA-GFP could be partially reverted to the behavior of Arf4-GFP by mutation of two aspartic acids in the HA tag to alanine. Our results, which indicate a high sensitivity of Arf4 to tagging, can explain the discrepancies between previous studies. We discuss the implications of this study for future work with tagged Arfs.
- Published
- 2020