Sorting signals required for trafficking of the cysteine-rich acidic repetitive transmembrane protein in Trypanosoma brucei.
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 micro1 adaptin, and mutations at amino acids essential for the function of the transport signal abolished the interaction of the signal with T. brucei micro1, strengthening the hypothesis of the involvement of the clathrin- and adaptor-dependent pathway in trafficking of CRAM via the FP.
Qiao
Xugang
X
Department of Pathology, New York University School of Medicine, 550 First Ave., New York, NY 10016, USA.
Chuang
Bin-Fay
BF
Jin
Yamei
Y
Muranjan
Madhavi
M
Hung
Chien-Hui
CH
Lee
Pei-Tseng
PT
Lee
Mary Gwo-Shu
MG
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D004705:Endocytosis; D005407:Flagella / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D021382:Protein Sorting Signals; D021381:Protein Transport / Q000502:physiology; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D014346:Trypanosoma brucei brucei / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D008565:Membrane Proteins; D021382:Protein Sorting Signals; D015800:Protozoan Proteins; C065453:cysteine-rich, acidic integral membrane protein, Trypanosoma brucei