The ADP-ribosylation factor-like 2 (ARL2) GTPase and its binding partner binder of ARL2 (BART) are ubiquitously expressed in rodent and human tissues and are most abundant in brain. Both ARL2 and BART are predominantly cytosolic, but a pool of each was found associated with mitochondria in a protease-resistant form. ARL2 was found to lack covalent N-myristoylation, present on all other members of the ARF family, thereby preserving the N-terminal amphipathic ␣-helix as a potential mitochondrial import sequence. An overlay assay was developed to identify binding partners for the BART⅐ARL2⅐GTP complex and revealed a specific interaction with a protein in bovine brain mitochondria. Purification and partial microsequencing identified the protein as an adenine nucleotide transporter (ANT). The overlay assay was performed on mitochondria isolated from five different tissues from either wild-type or transgenic mice deleted for ANT1. Results confirmed that ANT1 is the predominant binding partner for the BART⅐ARL2⅐GTP complex and that the structurally homologous ANT2 protein does not bind the complex. Cardiac and skeletal muscle mitochondria from ant1 Ϫ /ant1 Ϫ mice had increased levels of ARL2, relative to that seen in mitochondria from wild-type animals. We conclude that the amount of ARL2 in mitochondria is subject to regulation via an ANT1-sensitive pathway in muscle tissues.

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To examine the functions of the Arf-like protein, Arl1p, in Saccharomyces cerevisiae, a null allele, arl1 ::HIS3, was constructed in two strains. In one background only, loss of ARL1 resulted in temperature-sensitive (ts) growth (suppressed on high-osmolarity media). Allelic variation at the SSD1 locus accounted for differences between strains. Strains lacking ARL1 exhibited several defects in membrane traffic. First, arl1 strains secreted less protein as measured by TCA-precipitable radioactivity found in the media of [35 S ]-labelled cells. A portion of newly synthesized carboxypeptidase Y (CPY) was secreted rather than correctly targeted to the vacuole. Uptake of the fluidphase marker, lucifer yellow, was reduced. All these phenotypes were exacerbated in an ssd1 background. The ts phenotype of the arl1 ssd1 strain was suppressed by YPT1, the yeast Rab1a homologue, suggesting that ARL1 and YPT1 have partially overlapping functions. These findings demonstrate that ARL1 encodes a regulator of membrane traffic.

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