The LAMP‐like protein p67 plays an essential role in the lysosome of African trypanosomes

Abstract

SummaryRNAi knockdown was employed to study the function of p67, a lysosome-associated membrane protein (LAMP)-like type I transmembrane lysosomal glycoprotein in African trypanosomes. Conditional induction of p67 dsRNA resulted in specific~90% reductions in de novo p67 synthesis in both mammalian bloodstream and procyclic insect-stage parasites. Bloodstream cell growth was severely retarded with extensive death after > 24 h of induction. Biosynthetic trafficking of residual p67, and of the soluble lysosomal protease trypanopain, were unimpaired. Endocytosis of tomato lectin, a surrogate receptor-mediated cargo, was only mildly impaired (~20%), but proper lysosomal targeting was unaffected. p67 ablation had dramatic effects on lysosomal morphology with gross enlargement (four-to fivefold) and internal membrane profiles reminiscent of autophagic vacuoles. Ablation of p67 expression rendered bloodstream trypanosomes refractory to lysis by human trypanolytic factor (TLF), a lysosomally activated host innate immune mediator. Similar effects on lysosomal morphology and TLF sensitivity were also obtained by two pharmacological agents that neutralize lysosomal pH -chloroquine and bafilomycin A1. Surprisingly, however, lysosomal pH was not affected in ablated cells suggesting that other physiological alterations must account for increased resistance to TLF. These results indicate p67 plays an essential role in maintenance of normal lysosomal structure and physiology in bloodstream-stage African trypanosomes.

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

“…p67, a lysosomal associ-1 In our prior publications, we have used the term "trypanopain" to refer to TbbCATL as a generic name for this cathepsin L orthologue in all T. brucei subspecies. In accord with the recent proposal of Caffrey and Steverding (2009) Peck et al, 2008). TbSar1DN expression reduced the processing of gp100 to gp150, indicating a direct inhibition of ER exit ( Figure 1C, Tetϩ).…”

“…p67, a lysosomal associ-ated membrane protein-like type I transmembrane protein, is synthesized as a highly N-glycosylated 100-kDa ER glycoform (gp100). Poly-N-acetyllactosamine modification of the N-glycans in the Golgi results in formation of the 150-kDa glycoform (gp150) ( Figure 1C, TetϪ), which is subsequently degraded upon arrival in the lysosome (Alexander et al, 2002;Peck et al, 2008). TbSar1DN expression reduced the processing of gp100 to gp150, indicating a direct inhibition of ER exit ( Figure 1C, Tetϩ).…”

“…Rabbit anti-BiP, rabbit anti-VSG221, monoclonal mouse anti-p67 (mAb218), and rabbit anti-TbbCATL have all been described previously (Bangs et al, 1993;Alexander et al, 2002;Peck et al, 2008). Mouse anti-HA (Invitrogen, Carlsbad CA), rabbit anti-HA (Covance Research Products, Berkeley, CA), and mouse anti-Ty (UAB Hybridoma Facility, Birmingham, AL) were used as in previous publications (Sutterwala et al, 2008).…”

“…Pulse-chase metabolic radiolabeling with [ 35 S]methionine/cysteine of trypanosomes and immunoprecipitation of labeled reporters have been detailed previously (Triggs and Bangs, 2003;Peck et al, 2008). Unless stated otherwise, lysates for immunoprecipitation were prepared in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% deoxycholate, and 0.1% SDS).…”

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

“…The release of lysosomal proteases is a factor in the signature event of human infection, penetration of the central nervous system (36). Finally, lysosomal physiology is critical to the activity of an innate human serum resistance trait, trypanolytic factor, which limits the host range of Trypanosoma species (38).…”