title: Ubiquitination of the glycosomal matrix protein receptor PEX5 in Trypanosoma brucei by PEX4 displays novel features.
abstract: Trypanosomatids contain peroxisome-like organelles called glycosomes. Peroxisomal biogenesis involves a cytosolic receptor, PEX5, which, after its insertion into the organellar membrane, delivers proteins to the matrix. In yeasts and mammalian cells, transient PEX5 monoubiquitination at the membrane serves as the signal for its retrieval from the organelle for re-use. When its recycling is impaired, PEX5 is polyubiquitinated for proteasomal degradation. Stably monoubiquitinated TbPEX5 was detected in cytosolic fractions of Trypanosoma brucei, indicative for its role as physiological intermediate in receptor recycling. This modification's resistance to dithiothreitol suggests ubiquitin conjugation of a lysine residue. T. brucei PEX4, the functional homologue of the ubiquitin-conjugating (UBC) enzyme responsible for PEX5 monoubiquitination in yeast, was identified. It is associated with the cytosolic face of the glycosomal membrane, probably anchored by an identified putative TbPEX22. The involvement of TbPEX4 in TbPEX5 ubiquitination was demonstrated using procyclic ∆PEX4 trypanosomes. Surprisingly, glycosomal matrix protein import was only mildly affected in this mutant. Since other UBC homologues were upregulated, it might be possible that these have partially rescued PEX4's function in PEX5 ubiquitination. In addition, the altered expression of UBCs, notably of candidates involved in cell-cycle control, could be responsible for observed morphological and motility defects of the ∆PEX4 mutant.
authors.affiliation: Research Unit for Tropical Diseases, de Duve Institute, and Laboratory of Biochemistry, Université catholique de Louvain, Avenue Hippocrate 74, B-1200 Brussels, Belgium.
authors.forename: Melisa
authors.initials: M
authors.lastname: Gualdrón-López
authors.affiliation: Research Unit for Tropical Diseases, de Duve Institute, and Laboratory of Biochemistry, Université catholique de Louvain, Avenue Hippocrate 74, B-1200 Brussels, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Chevalier
authors.affiliation: Cell Biology Unit, de Duve Institute, Université catholique de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: Patrick
authors.initials: P
authors.lastname: Van Der Smissen
authors.affiliation: Cell Biology Unit, de Duve Institute, Université catholique de Louvain, Avenue Hippocrate 75, B-1200 Brussels, Belgium.
authors.forename: Pierre J
authors.initials: PJ
authors.lastname: Courtoy
authors.affiliation: Institute of Integrative Biology, University of Liverpool, Crown Street, Liverpool L69 7ZB, UK.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Rigden
authors.affiliation: Research Unit for Tropical Diseases, de Duve Institute, and Laboratory of Biochemistry, Université catholique de Louvain, Avenue Hippocrate 74, B-1200 Brussels, Belgium. Electronic address: paul.michels@ed.ac.uk.
authors.forename: Paul A M
authors.initials: PAM
authors.lastname: Michels
mesh_terms: D000818:Animals; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005786:Gene Expression Regulation; D055786:Gene Knockout Techniques; D007425:Intracellular Membranes / Q000378:metabolism; D008018:Life Cycle Stages; D008830:Microbodies / Q000378:metabolism*; D008954:Models, Biological; D021381:Protein Transport; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000648:ultrastructure; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D015800:Protozoan Proteins; D044763:Ubiquitin-Conjugating Enzymes
keywords: 4′,6-diamidino-2-phenylindole; DAPI; DTT; DUB; FBPase; GAPDH; GFP; Glycosome biogenesis; HXK; N-ethylmaleimide; NEM; ORF; PEX; PEX4; PEX5; PFK; PTS; Peroxin; TPI; TPR; Tet; Trypanosoma; UBC; Ubiquitination; deubiquitinating; dithiothreitol; fructose-1,6-bisphosphatase; glyceraldehyde-3-phosphate dehydrogenase; green-fluorescent protein; hexokinase; open‐reading frame; peroxin; peroxisomal-targeting signal; phosphofructokinase; tetracycline; tetratricopeptide repeat; triosephosphate isomerase; ubiquitin-conjugating