ABSTRACT
The endocytic pathway is of central importance for eukaryotic cells, as it enables uptake of extracellular materials, membrane protein quality control and recycling, as well as modulation of receptor signaling. While the ATPase p97 (VCP, Cdc48) has been found to be involved in the fusion of early endosomes and endolysosomal degradation, its role in endocytic trafficking is still incompletely characterized. Here, we identify myoferlin (MYOF), a ferlin family member with functions in membrane trafficking and repair, as a hitherto unknown p97 interactor. The interaction of MYOF with p97 depends on the cofactor PLAA previously linked to endosomal sorting. Besides PLAA, shared interactors of p97 and MYOF comprise several proteins involved in endosomal recycling pathways, including Rab11, Rab14, and the transferrin receptor CD71. Accordingly, a fraction of p97 and PLAA localizes to MYOF-, Rab11-, and Rab14-positive endosomal compartments. Pharmacological inhibition of p97 delays transferrin recycling, indicating that p97 promotes not only the lysosomal degradation, but also the recycling of endocytic cargo.
Subject(s)
Endosomes , Membrane Proteins , Biological Transport , Endosomes/metabolism , Membrane Proteins/metabolism , Protein Transport , Transferrin/metabolism , HumansABSTRACT
During terminal differentiation, the global protein complement is remodeled, as epitomized by erythrocytes, whose cytosol is ~98% globin. The erythroid proteome undergoes a rapid transition at the reticulocyte stage; however, the mechanisms driving programmed elimination of preexisting cytosolic proteins are unclear. We found that a mutation in the murine Ube2o gene, which encodes a ubiquitin-conjugating enzyme induced during erythropoiesis, results in anemia. Proteomic analysis suggested that UBE2O is a broad-spectrum ubiquitinating enzyme that remodels the erythroid proteome. In particular, ribosome elimination, a hallmark of reticulocyte differentiation, was defective in Ube2o-/- mutants. UBE2O recognized ribosomal proteins and other substrates directly, targeting them to proteasomes for degradation. Thus, in reticulocytes, the induction of ubiquitinating factors may drive the transition from a complex to a simple proteome.
Subject(s)
Erythroid Cells/cytology , Erythropoiesis/physiology , Ribosomal Proteins/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitination , Anemia/genetics , Anemia, Hypochromic/genetics , Animals , Erythrocytes/cytology , Erythrocytes/enzymology , Erythroid Cells/enzymology , Erythropoiesis/genetics , HEK293 Cells , Humans , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mutation , Proteasome Endopeptidase Complex/metabolism , Proteome/metabolism , Proteomics , Reticulocytes/cytology , Reticulocytes/enzymology , Ribosomes/metabolism , Ubiquitin-Conjugating Enzymes/genetics , beta-Globins/genetics , beta-Globins/metabolismABSTRACT
Protein misfolding and protein aggregation are causes of severe diseases as neurodegenerative disorders, diabetes and cancer. Therefore, the cell has to constantly monitor the folding status of its proteome. Chaperones and components of the ubiquitin-proteasome system are key players in the cellular protein quality control process. In order to characterize components of the protein quality control system in a well-established model eukaryote - the yeast Saccharomyces cerevisiae - we established new cytosolic model substrates based on firefly luciferase and ß-isopropylmalate dehydrogenase (Leu2). The use of these two different enzymes arranged in tandem as reporters enabled us to analyse the folding status and the degradation propensity of these new model substrates in yeast cells mutated in components of the cellular protein quality control system. The Hsp70 chaperone system known to be essential in the cellular protein quality control was chosen as a model for showing the high value of the luciferase-based model substrates in the characterization of components of the cytosolic protein quality control system in yeast.
