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Myristoylation is required for the intracellular localization and endocytic function of ARF6
1Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Abstract:
ADP-ribosylation factors (ARFs) are modified by a myristate group that is covalently linked to the second glycine residue at the amino terminus. With the recent evidence that ARF6 localizes to the cell periphery and plays a regulatory role in endocytic traffic, we have investigated the role of myristoylation on the membrane association, biological activity, and subcellular distribution of ARF6 in intact cells. A Gly2Ala mutation produced a nonmyristoylated protein that failed to associate with membranes, was cytosolic, and had no effect on endocytic transport. To determine if a different form of lipid modification could restore membrane association and biological activity, a nonmyristoylated ARF6 derivative was constructed that contained a prenyl group at the carboxy terminus. Prenylated ARF6 bound efficiently to membranes, but had no effect on receptor-mediated endocytosis and was mislocalized to distinct intracellular structures. Thus, although prenylation can replace myristoylation for membrane association, the latter appears to be critical for the proper targeting and biological activity of ARF6.
Insights
Myristoylation of ADP-ribosylation factor 6 (ARF6) is crucial for its membrane association and regulation of endocytic transport. Nonmyristoylated ARF6 is cytosolic and inactive, while prenylation restores membrane binding but not function.
Area of Science:
- Cell Biology
- Molecular Biology
- Protein Modification
Background:
- ADP-ribosylation factors (ARFs) are key regulators of intracellular membrane trafficking.
- ARF6 is known to localize to the cell periphery and influence endocytic pathways.
- Lipid modifications, such as myristoylation, are critical for ARF protein function.
Purpose of the Study:
- To investigate the specific role of myristoylation in ARF6 membrane association, biological activity, and subcellular localization.
- To compare the effects of myristoylation with other lipid modifications like prenylation on ARF6 function.
Main Methods:
- Site-directed mutagenesis to create nonmyristoylated ARF6 (Gly2Ala mutation).
- Construction of a prenylated ARF6 derivative.
- Analysis of membrane association, subcellular distribution, and endocytic transport in intact cells.
Main Results:
- The Gly2Ala mutation abolished ARF6 myristoylation, leading to cytosolic localization and loss of membrane association.
- Nonmyristoylated ARF6 failed to regulate endocytic transport.
- Prenylation of ARF6 restored membrane binding but resulted in mislocalization and no effect on endocytosis.
Conclusions:
- Myristoylation is essential for the proper membrane targeting and biological activity of ARF6.
- While prenylation can mediate membrane association, it cannot substitute for myristoylation in ARF6's role in endocytic traffic.
- This highlights the specific requirement of myristoylation for ARF6 function in regulating endocytosis.