Abstract
ARF-like 2 (ARL2) is a member of the ARF family and RAS superfamily of regulatory GTPases, predicted to be present in the last eukaryotic common ancestor, and essential in a number of model genetic systems. Though best studied as a regulator of tubulin folding, we previously demonstrated that ARL2 partially localizes to mitochondria. Here, we show that ARL2 is essential to a number of mitochondrial functions, including mitochondrial morphology, motility, and maintenance of ATP levels. We compare phenotypes resulting from ARL2 depletion and expression of dominant negative mutants and use these to demonstrate that the mitochondrial roles of ARL2 are distinct from its roles in tubulin folding. Testing of current models for ARL2 actions at mitochondria failed to support them. Rather, we found that knockdown of the ARL2 GTPase activating protein (GAP) ELMOD2 phenocopies two of three phenotypes of ARL2 siRNA, making it a likely effector for these actions. These results add new layers of complexity to ARL2 signaling, highlighting the need to deconvolve these different cell functions. We hypothesize that ARL2 plays essential roles inside mitochondria along with other cellular functions, at least in part to provide coupling of regulation between these essential cell processes.
Highlights
GTPases in the RAS superfamily have emerged as regulators of many specific signaling and metabolic pathways and provide integration between pathways through the use of common GTPases or effectors
Controls for specificity of knockdown include the sequence independence of the RNAs, mock transfected cells, and use of siRNAs directed against the closest ADP-ribosylation factor-like 2 (ARL2) paralog, ARL3, which lack each of the activities described here for ARL2
When we compared the microtubule staining profiles of mock transfected cells with those expressing ARL2 or ARL2[K71R], and those depleted for ARL2, we found that microtubule profiles were indistinguishable at all times examined (Figure 5; 48 hours); characterized by meshwork staining throughout the cytosol, Figure 5
Summary
GTPases in the RAS superfamily have emerged as regulators of many specific signaling and metabolic pathways and provide integration between pathways through the use of common GTPases or effectors. ARL2 is highly conserved in eukaryotes and ubiquitously expressed [1] It plays roles in both the regulation of tubulin folding and microtubule destruction [2,3], and is found in cytosol tightly bound to the tubulin specific co-chaperone, cofactor D, which shares those activities. Mutations in both ARL2 and cofactor D have been identified in a number of genetic screens linked to microtubules in model organisms that include S. cerevisiae, S. pombe, A. thaliana, and C. elegans [4,5,6,7,8,9]. We set out to examine its role(s) in mitochondria both to better understand its cellular functions and to provide potential insights into how these different essential cell roles may be integrated, as defects in any of the processes linked to ARL2 dysfunction are hallmarks of the transformed state, cancer, and a host of human diseases
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