Peroxisomal targeting signal-1 receptor protein PEX5 from Leishmania donovani. Molecular, biochemical, and immunocytochemical characterization.
The human pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other key metabolic pathways in unique subcellular microbodies called glycosomes, organelles related to the peroxisomes of mammals and yeast. The molecular machinery that carries out the specific targeting of glycosomal proteins to the organelle has not been characterized, although the bulk of glycosomal proteins contain the COOH-terminal tripeptide glycosomal peroxisomal targeting signal-1 (PTS-1) similar to the mammalian and fungal peroxisomal targeting signal. To characterize the mechanisms of glycosomal targeting, the gene encoding PEX5, designated LdPEX5, has been isolated from Leishmania donovani. LdPEX5 encodes a 625-amino acid protein with a molecular mass of 69.7 kDa. Like its homologs in yeast and humans, LdPEX5 predicts a protein with seven copies of a tetratricopeptide repeat in its COOH-terminal half proposed to mediate PTS-1 binding and three copies of a WXXX(Y/F) motif in its NH(2) terminus conjectured to be essential for protein translocation into the organelle. LdPEX5 was overexpressed in Escherichia coli and purified to homogeneity for binding experiments and generation of antibodies. Recombinant LdPEX5 bound xanthine phosphoribosyltransferase (XPRT), a PTS-1 containing glycosomal protein with a K(D) of 4.2 nm, but did not bind an XPRT in which the PTS-1 had been deleted. Moreover, binding studies with the COOH-terminal half of the LdPEX5 confirmed that this portion of the PEX5 protein was capable of binding the XPRT PTS-1 with an affinity of 17.3 nm. Confocal microsocopy revealed that LdPEX5 was predominantly in the cytosolic milieu, and genetic analysis implied that LdPEX5 was an essential gene.
Jardim
A
A
Department of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, Oregon 97201, USA.
Liu
W
W
Zheleznova
E
E
Ullman
B
B
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D006801:Humans; D007893:Leishmania donovani / Q000378:metabolism*; D008969:Molecular Sequence Data; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017421:Sequence Analysis
C000614264:PEX5 protein, human; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear