Using liposome- and column-based affinity chromatography, Arl5 was shown to interact with the GARP tethering complex; in Drosophila tissues GARP is partially displaced from the Golgi when Arl5 is absent, and in HeLa cells GARP becomes cytosolic on Arl5b depletion, establishing Arl5 as one of the factors that recruits GARP to the trans-Golgi in both flies and humans.

Arl5a and Arl5b localize to the trans-Golgi in mammalian cells; the constitutively active Arl5b(Q70L)-GFP mutant localizes efficiently to the Golgi whereas dominant-negative Arl5b(T30N)-GFP is dispersed in the cytoplasm; Arl5b depletion alters mannose-6-phosphate receptor distribution and reduces endosome-to-TGN transport of TGN38 and Shiga toxin.

ARFRP1 functions upstream of ARL1 and ARL5, which in turn recruit golgins and GARP respectively to the TGN; SYS1 regulates the ARFRP1-mediated recruitment of GARP; KO of ARL5 or ARFRP1 disperses the retrograde cargo TGN46 from the TGN, and GARP association with the TGN depends on the GTP-bound, active forms of ARL5 and ARFRP1.

Proximity biotinylation and interaction assays show ARMH3 (C10orf76) binds active but not inactive ARL5 and is recruited to the TGN in a SYS1-ARFRP1-ARL5-dependent manner; unlike GARP, ARMH3 is dispensable for retrograde cargo transport but activates PI4KB, accounting for the main pool of PI4P at the TGN.

Amino acids stimulate endosome-to-Golgi retrograde trafficking; Arl5 interacts with Ragulator in an amino acid-regulated manner, SLC38A9, v-ATPase and Ragulator (but not Rag GTPases or mTORC1) are essential for the stimulated trafficking, and Ragulator may act as a guanine nucleotide exchange factor to activate Arl5, which functions with its effector GARP.

Identifies ARL5A as an ARF-family small GTPase that acts as a molecular switch, cycling between inactive GDP-bound and active GTP-bound states, with GTP hydrolysis as its enzymatic activity and effector recruitment (GARP, ARMH3, PI4KB) as its biological output.

Documents predominant trans-Golgi network localization (strongest overlap with TGN46), dependent on the upstream ARFRP1-SYS1 complex, with a secondary endolysosomal pool that interacts with Ragulator in an amino acid-sensitive manner.

Establishes that ARL5-GARP-mediated endosome-to-TGN retrograde trafficking recycles TGN-resident cargo (TGN46, mannose-6-phosphate receptors, furin, sortilin), and that ARL5 depletion causes cargo mislocalization and endosomal accumulation.

Notes that no Xenopus tropicalis-specific functional studies were identified; conclusions rest on deep evolutionary conservation of ARL5 (present in LECA) and detailed mammalian ARL5A/ARL5B experiments.

Assigns the protein to the small GTPase superfamily, Arf family, with GTP-binding sites at residues 23-30, 69, and 125-128 and Mg(2+) binding at residues 30 and 47.