GTP-Binding-Defective ARL4D Alters Mitochondrial Morphology and Membrane Potential

Abstract

ARL4D, ARL4A, and ARL4C are closely related members of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of GTPases. All three ARL4 proteins contain nuclear localization signals (NLSs) at their C-termini and are primarily found at the plasma membrane, but they are also present in the nucleus and cytoplasm. ARF function and localization depends on their controlled binding and hydrolysis of GTP. Here we show that GTP-binding-defective ARL4D is targeted to the mitochondria, where it affects mitochondrial morphology and function. We found that a portion of endogenous ARL4D and the GTP-binding-defective ARL4D mutant ARL4D(T35N) reside in the mitochondria. The N-terminal myristoylation of ARL4D(T35N) was required for its localization to mitochondria. The localization of ARL4D(T35N) to the mitochondria reduced the mitochondrial membrane potential (ΔΨm) and caused mitochondrial fragmentation. Furthermore, the C-terminal NLS region of ARL4D(T35N) was required for its effect on the mitochondria. This study is the first to demonstrate that the dysfunctional GTP-binding-defective ARL4D is targeted to mitochondria, where it subsequently alters mitochondrial morphology and membrane potential.

The ARL2 GTPase regulates mitochondrial fusion from the intermembrane space

“…30,33,34,39 There are several other proteins with roles outside of mitochondria acting on different pathways inside mitochondria, including STAT3, 76,77 MEF2D, 78 fumarase, 79 several DNA repair proteins, 80 and others. 79,[81][82][83][84][85] Nevertheless, when studying a protein with multiple cellular roles (the term "moonlighting roles" is increasingly used), it is important to resolve the different actions to allow functional and mechanistic studies of each. This can be particularly challenging when essential cellular functions are involved or when the 2 actions may yield similar phenotypes.…”

Mitochondria, calcium, and tumor suppressor Fus1: At the crossroad of cancer, inflammation, and autoimmunity

“…Recently, several myristoylated proteins residing in mitochondria have been described. Among them are proteins of the inner and outer mitochondrial membranes: GTPase ARL4D [ 96 ], ChChd3 [ 97 ], NADH-cytochrome b5 reductase [ 98 , 99 ], truncated forms of actin [ 100 ], and Bid [ 101 ]. However, Fus1 is the first putative member of the Ca 2+ /myristoyl switch protein family localized to mitochondria.…”

Higher order signaling: ARL2 as regulator of both mitochondrial fusion and microtubule dynamics allows integration of 2 essential cell functions

“…57 ARL2 is among the list of GTPases with essential regulatory functions acting in or on mitochondria, that currently includes the well-established regulators OPA1, MFNs, MIROs, and DRP1, and recent addition of ARL4D. [58][59][60] The findings that ARL2 regulates 2 such fundamental and ancient aspects of eukaryotic biology is quite striking but consistent with its predicted presence at the origin of eukaryotes, its ubiquity, and high degree of sequence conservation. The stable (at least through several days of purification in vitro) association of ARL2 with TBCD and low levels of monomer in the cytosol is expected to limit the availability of ARL2 for import into mitochondria.…”