Functional Expression and Characterization of Human Myristoylated-Arf1 in Nanodisc Membrane Mimetics.

Abstract

Lipidated small GTP-binding proteins of the Arf family interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Here, we focus on the ADP-ribosylation factor 1 (Arf1), which interacts with numerous proteins in the Arf pathway, such as the ArfGAP ASAP1 that is highly expressed and activated in several cancer cell lines and associated with enhanced migration, invasiveness, and poor prognosis. Understanding the molecular and mechanistic details of Arf1 regulation at the membrane via structural and biophysical studies requires large quantities of fully functional protein bound to lipid bilayers. Here, we report on the production of a functional human Arf1 membrane platform on nanodiscs for biophysical studies. Large scale bacterial production of highly pure, N-myristoylated human Arf1 has been achieved, including complex isotopic labeling for nuclear magnetic resonance (NMR) studies, and the myr-Arf1 can be readily assembled in small nanoscale lipid bilayers (nanodiscs, NDs). It is determined that myr-Arf1 requires a minimum binding surface in the NDs of ∼20 lipids. Fluorescence and NMR were used to establish nucleotide exchange and ArfGAP-stimulated GTP hydrolysis at the membrane, indicating that phophoinositide stimulation of the activity of the ArfGAP ASAP1 is ≥2000-fold. Differences in nonhydrolyzable GTP analogues are observed, and GMPPCP is found to be the most stable. Combined, these observations establish a functional environment for biophysical studies of Arf1 effectors and interactions at the membrane.

• Authors: Yifei Li, Olivier Soubias, Jess Li, Shangjin Sun, Paul A Randazzo, R Andrew Byrd
• Journal: Biochemistry (Biochemistry) Vol. 58 Issue 10 Pg. 1423-1431 (03 12 2019) ISSN: 1520-4995 [Electronic] United States
• PMID: 30735034 (Publication Type: Journal Article, Research Support, N.I.H., Intramural)

Chemical References

• Lipid Bilayers
• Membrane Proteins
• Myristic Acid
• ADP-Ribosylation Factor 1
• ADP-Ribosylation Factors
• ARF1 protein, human

Topics

• ADP-Ribosylation Factor 1 (chemistry, genetics, metabolism)
• ADP-Ribosylation Factors (metabolism)
• Humans
• Intracellular Membranes (chemistry, metabolism)
• Lipid Bilayers (chemistry)
• Membrane Proteins (genetics, metabolism)
• Membranes (chemistry, metabolism)
• Myristic Acid (metabolism)