Multiple Motifs Regulate Trafficking of the LAMP‐Like Protein p67 in the Ancient Eukaryote Trypanosoma brucei

Abstract

p67 is a lysosome-associated membrane protein-like lysosomal type I transmembrane glycoprotein in African trypanosomes. The p67 cytoplasmic domain (CD) is both necessary and sufficient for lysosomal targeting in procyclic insect-stage parasites. The p67CD contains two [DE]XXXL[LI]-type dileucine motifs, which function as lysosomal targeting signals in mammalian cells. Using a green fluorescent protein fusion to the p67 transmembrane and cytoplasmic domains as a reporter system, we investigated the role of these motifs in lysosomal targeting in procyclic trypanosomes. Pulse-chase turnover studies, steady-state immunolocalization and quantitative flow cytometry all gave consistent results. Mutagenesis of the membrane-distal dileucine motif impairs lysosomal trafficking leading to partial appearance of the reporter on the cell surface. Mutagenesis of the membrane-proximal motif has little effect on proper targeting. Simultaneous mutagenesis of both motifs results in quantitative delivery to the cell surface. Thus, the distal motif plays a dominant role, but both dileucine motifs are necessary for maximal lysosomal targeting. Additional studies suggest that the upstream acidic residues in each motif influence lysosomal targeting and may also affect forward trafficking in the early secretory pathway. These results strongly suggest an evolutionary conservation in lysosomal trafficking mechanisms in the ancient eukaryote Trypanosoma brucei.

Role of AP-1 in Developmentally Regulated Lysosomal Trafficking in Trypanosoma brucei

“…In procyclic insect stage (PCF) trypanosomes, the cytoplasmic domain is both necessary and sufficient for lysosomal targeting of a heterologous reporter, and its deletion results in mistargeting of p67 to the cell surface (1). The cytoplasmic domain contains two canonical dileucine motifs, mutation of which also results in delivery to the cell surface (47). These findings strongly indicate the existence of cognate cytoplasmic machinery for lysosomal delivery of p67 in PCF trypanosomes.…”

“…Standard propagation of the cultured PCF and BSF Lister 427 strain T. brucei brucei in Cunningham's and HMI9 media (15, 23) is described elsewhere (38,47). The tetracycline-responsive 29-13 PCF and 13-90 BSF derivatives of Lister 427 were used for all experiments (50).…”

“…Having previously studied the innate signals involved in p67 targeting (1,47), we now turned our attention to the cognate machinery for post-Golgi apparatus sorting. Specifically, we investigate the role of trypanosomal AP-1 in stage-specific biosynthetic trafficking to the lysosome using RNA interference (RNAi)-mediated silencing of the Tb1 (geneDB no.…”

Streamlined Architecture and Glycosylphosphatidylinositol-dependent Trafficking in the Early Secretory Pathway of African Trypanosomes

“…All immunoprecipitates were sepa-E. S. Sevova and J. D. Bangs Molecular Biology of the Cell 4740 rated on 12% SDS-polyacrylamide gel electrophoresis (PAGE) gels and visualized via the Typhoon system (GE Healthcare). Quantification of phosphorimages was done by ImageQuant software as described previously (Tazeh and Bangs, 2007).…”

“…All immunoprecipitates were sepa-E. S. Sevova and J. D. Bangs Molecular Biology of the Cell 4740 rated on 12% SDS-polyacrylamide gel electrophoresis (PAGE) gels and visualized via the Typhoon system (GE Healthcare). Quantification of phosphorimages was done by ImageQuant software as described previously (Tazeh and Bangs, 2007).Sequential immunoprecipitation experiments have been detailed previously (Bangs et al, 1996). In brief, parasites were radiolabeled with [ 35 S]methionine/cysteine for 2.5 h. Cells were lysed in TEN buffer (50 mM Tris-HCl, 150 mM NaCl, and 5 mM EDTA, pH 7.5) with 0.5% NP-40, protease inhibitor cocktail (2 g/ml each of leupeptin, antipain, pepstatin, and chymostatin), 0.1 mM tosyllysine chloromethyl ketone, and 0.5 mM phenylmethylsulfonyl fluoride.…”