ARL4D is a developmentally regulated member of the ADP-ribosylation factor/ARF-like protein (ARF/ARL) family of Ras-related GTPases. Although the primary structure of ARL4D is very similar to that of other ARF/ARL molecules, its function remains unclear. Cytohesin-2/ARF nucleotide-binding-site opener (ARNO) is a guanine nucleotide-exchange factor (GEF) for ARF, and, at the plasma membrane, it can activate ARF6 to regulate actin reorganization and membrane ruffling. We show here that ARL4D interacts with the C-terminal pleckstrin homology (PH) and polybasic c domains of cytohesin-2/ARNO in a GTP-dependent manner. Localization of ARL4D at the plasma membrane is GTP-and N-terminal myristoylation-dependent. ARL4D(Q80L), a putative active form of ARL4D, induced accumulation of cytohesin-2/ARNO at the plasma membrane. Consistent with a known action of cytohesin-2/ARNO, ARL4D(Q80L) increased GTP-bound ARF6 and induced disassembly of actin stress fibers. Expression of inactive cytohesin-2/ARNO(E156K) or small interfering RNA knockdown of cytohesin-2/ARNO blocked ARL4D-mediated disassembly of actin stress fibers. Similar to the results with cytohesin-2/ARNO or ARF6, reduction of ARL4D suppressed cell migration activity. Furthermore, ARL4D-induced translocation of cytohesin-2/ARNO did not require phosphoinositide 3-kinase activation. Together, these data demonstrate that ARL4D acts as a novel upstream regulator of cytohesin-2/ARNO to promote ARF6 activation and modulate actin remodeling. INTRODUCTIONADP-ribosylation factors (ARFs) are small GTPases involved in membrane transport, maintenance of organelle integrity, and activation of phospholipase D and phosphatidylinositol 4-phosphate 5-kinase Chavrier and Goud, 1999;Takai et al., 2001;D'Souza-Schorey and Chavrier, 2006). ARF1 is mainly associated with the Golgi apparatus, and it regulates vesicle budding of transport events (Stearns et al., 1990;Balch et al., 1992). ARF6 can regulate peripheral membrane dynamics and actin rearrangements at the plasma membrane (Donaldson, 2003;Sabe, 2003) such as stress fibers disassembly (D'Souza-Schorey et al., 1997;Boshans et al., 2000), formation of plasma membrane protrusions and ruffles (Radhakrishna et al., 1996;D'Souza-Schorey et al., 1997;Franco et al., 1999), cell migration (Palacios et al., 2001;Santy and Casanova, 2001), cell adhesion (Palacios et al., 2001), and regulation of endosomal membrane traffic (D'Souza-Schorey et al., 1995;Radhakrishna and Donaldson, 1997). Similar to other guanosine triphosphate (GTP)-binding proteins, ARF function depends on the highly controlled binding and hydrolysis of GTP. The conformational changes that accompany the binding of GDP or GTP can lead directly to changes in the affinity of the GTPase for proteins, lipids, and membranes. Interconversion between the two states of ARFs is most likely achieved through guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) Donaldson and Jackson, 2000;Jackson and Casanova, 2000).ARF-GEFs are linked to vesicular trafficking and ...

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.

ADP-ribosylation factor (Arf)-like 4D (Arl4D), one of the Arf-like small GTPases, functions in the regulation of cell morphology, cell migration, and actin cytoskeleton remodeling. EB1 is a microtubule plus-end tracking protein that preferentially localizes at the tips of the plus ends of growing microtubules and at the centrosome. EB1 depletion results in many centrosome-related defects. Here, we report that Arl4D promotes the recruitment of EB1 to the centrosome and regulates microtubule nucleation. We first showed that Arl4D interacts with EB1 in a GTP-dependent manner. This interaction is dependent on the C-terminal EBH region of EB1 and partially dependent on an SxLP motif of Arl4D. We found that Arl4D colocalized with γ-tubulin in centrosomes and the depletion of Arl4D resulted in a centrosomal microtubule nucleation defect. We further demonstrated that abolishing Arl4D-EB1 interaction decreased microtubule nucleation rate and diminished the centrosomal recruitment of EB1 without affecting MT growth rate. In addition, Arl4D binding to EB1 increased the association between the p150 subunit of dynactin and EB1, which is important for microtubule stabilization. Together, our results indicate that Arl4D modulates microtubule nucleation through regulation of the EB1-p150 association at the centrosome. [Media: see text]