Ubiquitylation is required for degradation of transmembrane surface proteins in trypanosomes.
The surface of Trypanosoma brucei is dominated by glycosyl-phosphatidylinositol (GPI)-anchored proteins, and endocytosis is clathrin dependent. The vast majority of internalized GPI-anchored protein is efficiently recycled, while the processes by which transmembrane domain (TMD) proteins are internalized and sorted are unknown. We demonstrate that internalization of invariant surface glycoprotein (ISG)65, a trypanosome TMD protein, involves ubiquitylation and also requires clathrin. We find a hierarchical requirement for cytoplasmic lysine residues in internalization and turnover, and a single position-specific lysine is sufficient for degradation, surface removal and attachment of oligoubiquitin chains. Ubiquitylation is context dependent as provision of additional lysine residues by C-terminal fusion of neuronal precursor cell-expressed developmentally downregulated protein (NEDD)8 fails to support ubiquitylation. Attachment of NEDD8 leads to degradation by a second ubiquitin-independent pathway. Moreover, degradation of ubiquitylated or NEDDylated substrate takes place in an acidic compartment and is proteosome independent. Significantly, in non-opisthokont lineages, Rsp5p or c-Cbl, the E3 ubiquitin ligases acting on endocytic cargo, are absent but Uba1 class genes are present and are required for cell viability and ISG65 ubiquitylation. Hence, ubiquitylation is an evolutionarily conserved mechanism for internalization of surface proteins, but aspects of the machinery differ substantially between the major eukaryotic lineages.
Chung
Wei-Lian
WL
Department of Pathology, University of Cambridge, Cambridge, UK.
Leung
Ka Fai
KF
Carrington
Mark
M
Field
Mark C
MC
D000818:Animals; D002966:Clathrin / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009088:Mucoproteins / Q000378:metabolism; D021381:Protein Transport; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D014346:Trypanosoma brucei brucei / Q000378:metabolism* / Q000502:physiology; D025801:Ubiquitin / Q000378:metabolism*
D002966:Clathrin; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D009088:Mucoproteins; D015800:Protozoan Proteins; D025801:Ubiquitin; C010393:lysin, gastropoda