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Eukaryotic Cell, September 2009, p. 1352-1361, Vol. 8, No. 9
1535-9778/09/$08.00+0     doi:10.1128/EC.00156-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Ngii N. Tazeh,{dagger} Jason S. Silverman, Kevin J. Schwartz, Elitza S. Sevova, Shaheen S. Sutterwala, and James D. Bangs*

Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, 1550 Linden Drive, Madison, Wisconsin 53706

Received 1 June 2009/ Accepted 24 June 2009

African trypanosomes are the causative agents of human trypanosomiasis (sleeping sickness). The pathogenic stage of the parasite has unique adaptations to life in the bloodstream of the mammalian host, including upregulation of endocytic and lysosomal activities. We investigated stage-specific requirements for cytoplasmic adaptor/clathrin machinery in post-Golgi apparatus biosynthetic sorting to the lysosome using RNA interference silencing of the Tbµ1 subunit of adaptor complex 1 (AP-1), in conjunction with immunolocalization, kinetic analyses of reporter transport, and quantitative endocytosis assays. Tbµ1 silencing was lethal in both stages, indicating a critical function(s) for the AP-1 machinery. Transport of soluble and membrane-bound secretory cargoes was Tbµ1 independent in both stages. In procyclic parasites, trafficking of the lysosomal membrane protein, p67, was disrupted, leading to cell surface mislocalization. The lysosomal protease trypanopain was also secreted, suggesting a transmembrane-sorting receptor for this soluble hydrolase. In bloodstream trypanosomes, both p67 and trypanopain trafficking were unaffected by Tbµ1 silencing, suggesting that AP-1 is not necessary for biosynthetic lysosomal trafficking. Endocytosis in bloodstream cells was also unaffected, indicating that AP-1 does not function at the flagellar pocket. These results indicate that post-Golgi apparatus sorting to the lysosome is critically dependent on the AP-1/clathrin machinery in procyclic trypanosomes but that this machinery is not necessary in bloodstream parasites. We propose a simple model for stage-specific default secretory trafficking in trypanosomes that is consistent with the behavior of other soluble and glycosylphosphatidylinositol-anchored cargos and which is influenced by upregulation of endocytosis in bloodstream parasites as an adaptation to life in the mammalian bloodstream.

* Corresponding author. Mailing address: Department of Medical Microbiology and Immunology, University of Wisconsin School of Medicine and Public Health, 1550 Linden Drive, Madison, WI 53706. Phone: (608) 262-3110. Fax: (608) 262-8418. E-mail: jdbangs{at}wisc.edu

{triangledown} Published ahead of print on 6 July 2009.

{dagger} Present address: Carver College of Medicine, 200 CMAB, Iowa City, IA 52242.

Eukaryotic Cell, September 2009, p. 1352-1361, Vol. 8, No. 9
1535-9778/09/$08.00+0     doi:10.1128/EC.00156-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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