Identification and characterization of YlPex5p, a component of the peroxisomal translocation machinery of Yarrowia lipolytica

Peroxisomes are ubiquitous eukaryotic organelles. PEX genes encode peroxins (proteins required for peroxisome assembly). A genetic screen identified mutants of the yeast Yarrowia lipolytica that fail to assemble functional peroxisomes. One strain, pex5-1, has abnormally small, clustered peroxisomes that are often surrounded by membranous material. The PEX5 gene encodes YlPex5p. Pex5 proteins have been shown to function as receptors for type I peroxisomal targeting signals (PTS1s) in other species, interacting with PTS1s through their characteristic tetratricopeptide (TPR) domain. Biochemical characterization of the mutants pex5-1 and pex5-KO (a PEX5 gene disruption strain) showed that YlPex5p is a component of the peroxisomal translocation machinery. Mutations in PEX5 prevent the translocation into the matrix of most proteins successfully targeted to peroxisomes. These proteins, including a 62-kD polypeptide (p62) recognized by antibodies to a PTS1 (SKL), seem to be trapped in the peroxisomal membrane at an intermediate stage of translocation in pex5 mutants. The evidence presented suggests that there are at least two distinct translocation machineries for peroxisomal protein import. YlPex5p is intraperoxisomal. In wild-type peroxisomes, YlPex5p is associated primarily with the inner surface of the peroxisomal membrane, but approximately one-third of YlPex5p is localized to the peroxisomal matrix. Most YlPex5p in the matrix is complexed with two anti-SKL-reactive polypeptides: p62, and a 64-kD protein (p64). However, in pex5-1 cells, peroxisomal Pex5 protein is localized exclusively to the matrix and forms no complex with the membrane-associated p62. The nature of the association of YlPex5p with PTS1 signals was investigated with in vitro binding assays. A recombinant YlPex5p fusion protein interacted specifically, directly and autonomously with a protein terminating in a PTS1. In vitro translated wild-type YlPex5p specifically recognized functional PTS1s. This activity is abrogated by a glycine-to-aspartic acid substitution at a conserved residue in the TPR domain (G455D) of the protein encoded by the pex5-1 allele. Deletion analysis demonstrated that an intact TPR domain of YlPex5p is necessary, but not sufficient, for both interaction with a PTS1 and functional complementation of the pex5-KO strain. A model for YlPex5p action is presented.