THE PAL1 GENE-PRODUCT IS A PEROXISOMAL ATP-BINDING CASSETTE TRANSPORTER IN THE YEAST SACCHAROMYCES-CEREVISIAE

The PAL1 gene was isolated using PCR and degenerate oligonucleotide pr imers corresponding to highly conserved amino acid sequence motifs dia gnostic of the ATP-binding cassette domain of the superfamily of membr ane-bound transport proteins typified by mammalian multidrug resistanc e transporter 1 and Saccharomyces cerevisiae Ste6. The deduced PAL1 ge ne product is similar in length to, has the same predicted topology as , and shares the highest degree of amino acid sequence identity with t wo human proteins, adrenoleukodystrophy protein and peroxisomal membra ne protein (70 kD), which are both presumptive ATP-binding cassette tr ansporters thought to be constituents of the peroxisomal membrane. As judged by hybridization of a PAL1 probe to isolated RNA and by express ion of a PAL1-lacZ fusion, a PAL1 transcript was only detectable when cells were grown on oleic acid, a carbon source which requires the bio genesis of functional peroxisomes for its metabolism. A pal1 Delta mut ant grew normally on either glucose- or glycerol-containing media; how ever, unlike PAL1(+) cells (or the pal1 Delta mutant carrying the PAL1 gene on a plasmid), pal1 Delta cells were unable to grow on either a solid medium or a liquid medium containing oleic acid as the sole carb on source. Antibodies raised against a chimeric protein in which the C OOH-terminal domain of Pal1 was fused to glutathione S-transferase spe cifically recognized a protein in extracts from wild-type cells only w hen grown on oleic acid; this species represents the PAL1 gene product because it was missing in pal1 Delta cells and more abundant in pal1 Delta cells expressing PAL1 from a multicopy plasmid. The Pal1 polypep tide was highly enriched in the organellar pellet fraction prepared fr om wild-type cells by differential centrifugation and comigrated upon velocity sedimentation in a Nycodenz gradient with a known component o f the peroxisomal matrix, 3-oxoacyl-CoA thiolase. As judged by both su bcellular fractionation and indirect immunofluorescence, localization of 3-oxoacyl-CoA thiolase to peroxisomes was unchanged whether Pal1 wa s present, absent, or overexpressed. These findings demonstrate that P al1 is a peroxisome-specific protein, that it is required for peroxiso me function, but that it is not necessary for the biogenesis of peroxi somes or for the import of 3-oxoacyl-CoA thiolase (and at least two ot her peroxisomal matrix proteins).