ARL4, an ARF-like protein that is developmentally regulated and localized to nuclei and nucleoli

ADP-ribosylation factors (ARFs) are highly conserved similar to 20-kDa guan ine nucleotide-binding proteins that participate in both exocytic and endoc ytic vesicular transport pathways via mechanisms that are only partially un derstood. Although several ARF-like proteins (ARLs) are known, their biolog ical functions remain unclear. To characterize its molecular properties, we cloned mouse and human ARL4 (mARL4 and hARL4) cDNA. The appearance of mous e ARL4 mRNA during embryonic development coincided temporally with the sequ ential formation of somites and the establishment of brain compartmentation . Using ARL4-specific antibody for immunofluorescence microscopy, we observ ed that endogenous mARL4 in cultured Sertoli and neuroblastoma cells was ma inly concentrated in nuclei. When expressed in COS7 cells, ARL4-T34N mutant , predicted to exist with GDP bound, was concentrated in nucleoli. Yeast tw o-hybrid screening and in vitro protein-interaction assays showed that hARL 4 interacted with importin-alpha through its C-terminal NLS region and that the interaction was not nucleotide-dependent. Like ARL2 and -3, recombinan t hARL4 did not enhance cholera toxin-catalyzed auto-ADP-ribosylation. Its binding of guanosine 5'-O-(thiotriphosphate) was modified by phospholipid a nd detergent, and the N terminus of hARL4, like that of ARF, was myristoyla ted. Our findings suggest that ARL4, with its distinctive nuclear/nucleolar localization and pattern of developmental expression, may play a unique ro le(s) in neurogenesis and somitogenesis during embryonic development and in the early stages of spermatogenesis in adults.