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Eukaryotic Cell, August 2006, p. 1229-1242, Vol. 5, No. 8
1535-9778/06/$08.00+0     doi:10.1128/EC.00064-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Sorting Signals Required for Trafficking of the Cysteine-Rich Acidic Repetitive Transmembrane Protein in Trypanosoma brucei

Xugang Qiao, Bin-Fay Chuang, Yamei Jin, Madhavi Muranjan, Chien-Hui Hung, Pei-Tseng Lee, and Mary Gwo-Shu Lee^*

Department of Pathology, New York University School of Medicine, 550 First Ave., New York, New York 10016

Received 2 March 2006/ Accepted 12 May 2006

In trypanosomatids, endocytosis and exocytosis are restricted to the flagellar pocket (FP). The cysteine-rich acidic repetitive transmembrane (CRAM) protein is located at the FP of Trypanosoma brucei and potentially functions as a receptor or an essential component for lipoprotein uptake. We characterized sorting determinants involved in efficient trafficking of CRAM to and from the FP of T. brucei. Previous studies indicated the presence of signals in the CRAM C terminus, specific for its localization to the FP and for efficient endocytosis (H. Yang, D. G. Russell, B. Zeng, M. Eiki, and M.G.-S. Lee, Mol. Cell. Biol. 20:5149-5163, 2000.) To delineate functional domains of putative sorting signals, we performed a mutagenesis series of the CRAM C terminus. Subcellular localization of CRAM mutants demonstrated that the amino acid sequence between –5 and –14 (referred to as a transport signal) is essential for exporting CRAM from the endoplasmic reticulum to the FP, and mutations of amino acids at –12 (V), –10 (V), or –5 (D) led to retention of CRAM in the endoplasmic reticulum. Comparison of the endocytosis efficiency of CRAM mutants demonstrated that the sequence from amino acid –5 to –23 (referred to as a putative endocytosis signal) is required for efficient endocytosis and overlaps with the transport signal. Apparently the CRAM-derived sorting signal can efficiently interact with the T. brucei µ1 adaptin, and mutations at amino acids essential for the function of the transport signal abolished the interaction of the signal with T. brucei µ1, strengthening the hypothesis of the involvement of the clathrin- and adaptor-dependent pathway in trafficking of CRAM via the FP.

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* Corresponding author. Mailing address: New York University School of Medicine, Department of Pathology, 550 First Ave., New York, NY 10016. Phone: (212) 263-8260. Fax: (212) 263-8179. E-mail: leeg02{at}med.nyu.edu.

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Eukaryotic Cell, August 2006, p. 1229-1242, Vol. 5, No. 8
1535-9778/06/$08.00+0     doi:10.1128/EC.00064-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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