Peroxisomal targeting protein 14 (PEX14) from Leishmania donovani. Molecular, biochemical, and immunocytochemical characterization.
Pathogens of the Leishmania and Trypanosoma genera compartmentalize glycolytic and other nutritional pathways in glycosomes, unique subcellular organelles related to the peroxisomes of mammals and yeasts. Most glycosomal proteins are targeted to the glycosomes by a COOH-terminal tripeptide signal similar to the peroxisomal targeting signal-1 (PTS-1). It has been proposed that PTS-1 forms a complex with the PEX5 receptor protein which then docks to the glycosomal membrane through interactions with the membrane associated PEX14 protein. To analyze the role of PEX14 in glycosomal protein import, the gene encoding the L. donovani PEX14 (LdPEX14) was isolated and shown to encode a 464 amino acid protein that exhibited very limited sequence homology with peroxisomal PEX14 proteins. In vitro binding experiments with purified recombinant LdPEX14 and LdPEX5 confirmed that LdPEX14-LdPEX5 interacted with a K(d) of 2.75 microM. When LdPEX5 was preloaded with a PTS-1 peptide, the affinity of the LdPEX14-LdPEX5 interaction affinity increased. Furthermore, binding experiments with truncated forms of LdPEX5 and LdPEX14 showed that the interaction domains localized to the amino terminal region of both proteins. Finally, confocal microscopy, subcellular fractionation, and differential extraction experiments indicated that LdPEX14 is a soluble protein that associates tightly with the glycosomal membrane and further support the role of LdPEX14 in forming a docking complex involved in glycosome biogenesis.
Jardim
Armando
A
Institute of Parasitology, Macdonald Campus of McGill University, 21, 111 Lakeshore Road, Ste. Anne-de-Bellevue, Canada. armando.jardim@mcgill.ca
Rager
Nicolle
N
Liu
Wei
W
Ullman
Buddy
B
D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D003001:Cloning, Molecular; D015151:Immunoblotting; D007893:Leishmania donovani / Q000235:genetics* / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D018160:Receptors, Cytoplasmic and Nuclear / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013347:Subcellular Fractions / Q000378:metabolism
C470663:PEX14 protein, Leishmania donovani; D000074435:Peroxisome-Targeting Signal 1 Receptor; D015800:Protozoan Proteins; D018160:Receptors, Cytoplasmic and Nuclear