Mapping of the functional domains of TOR, an adaptor protein regulating protein trafficking in Leishmania.
While investigating how Leishmania becomes resistant to toxic purine nucleosides, we identified and cloned the TOR gene [1,2]. TOR is ah atypically multidrug resistance protein that was originally discovered by its ability, when present in high levels, to render Leishmania resistant to toxic nucleosides. Subsequent research showed that it also elicited resistance to a wide variety of structurally diverse toxins, among them many of the clinically important anti-Leishmania compounds. TOR is found in at least two regions in Leishmania: the mitochondria and the Golgi/trans Golgi network. In the Golgi/trans Golgi network, TOR acts as a traffic regulator and influences the amount of purine transporters which reach the plasma membrane .
Truncation mutants expressed in Leishmania and the yeast Saccharomyces and far western blots showed that the minimal domain extending from Met31 to Ser204 was responsible for interacting with TOTs (i.e., targets of TOR). Expression of the C terminal half of TOR as a "quasi" dominant-negative mutant showed that it also exhibited biological activity. Deletion of a putative clathrin binding domain in the C terminal half by site specific mutagenesis abolished TOR's ability to internalize the adenosine transporter. Far western experiments showed that the C terminal half of TOR also interacted with several of the Rab proteins. Furthermore, high level ectopic expression of these Rab proteins in Leishmania acted as a dominant-negative mutant and impaired the ability of TOR to internalize the trafficking of the adenosine permease. Saccharomyces Rab4 and Rab5 null mutants were no longer able to support the intemalization of the Leishmania adenosine permease by TOR.
These data indicate that TOR is an adaptor protein interacting with TOTs at one end and multiple components of the vesicle trafficking apparatus at the other end. TOR appears to be the progenitor of a new class of endocytotic accessory proteins. In contrast to the other heterotetrameric and monomeric adaptor protein classes, TOR is a homotetrameric protein.
[1.] Detke, S. (1997) Identification of a transcription factor like protein at the TOR locus in Leishmania mexicana amazonensis. Mol. Biochem. Parasitol. 90:505-511.
[2.] Kerby, B.R., and Detke, S. (1993) Reduced purine accumulation is encoded on an amplified DNA in Leishmania mexicana amazonensis resistant to toxic nucleosides. Mol. Biochem. Parasitol. 60:171-185.
[3.] Detke, S. (2007) TOR-induced resistance to toxic adenosine analogs in Leishmania brought about by the internalization and degradation of the adenosine permease. Exp. Cell Res. 313:1963-1978.
Department of Biochemistry & Molecular Biology, University of North Dakota, Grand Forks, North Dakota
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|Title Annotation:||COMMUNICATIONS--GRADUATE; target of rapamycin|
|Publication:||Proceedings of the North Dakota Academy of Science|
|Date:||Apr 1, 2009|
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