†These authors contributed equally to this work.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.

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Secreted aspartyl proteinases (Saps) from Candida albicans are encoded by a multi-gene family and are considered to be putative virulence factors for candidiasis. SAP4-6 mRNAs were first detected during hyphae formation and were assumed to play roles in the development of disseminated candidiasis. Recombinant Sap proteins (Sap2-6) were prepared and specific antibodies were generated against Sap2-6. The presence of Sap4, Sap5 and Sap6, but not Sap2 or Sap3, was demonstrated in culture supernatants of C. albicans after induction of hyphae formation. In parallel to hyphae formation, Sap5 ("40 kDa) was detected as early as "6 h after induction at neutral pH, and Sap4/6 ("43 kDa) were detected after "24 h when the culture medium became acidic. The differential secretion of Sap5 and Sap4/6 was affected when the culture medium pH was buffered at pH 65 or pH 45. In addition, intracellular pools of Sap4-6 seem to exist, and protein is not necessary for Sap4-6 induction. This study provides the first evidence that Sap4-6 proteins in C. albicans are differentially produced and secreted during hyphae formation.