EC Accepts, published online ahead of print on 9 October 2009

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Eukaryotic Cell doi:10.1128/EC.00169-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Heterologous expression studies in yeast reveal two distinct trypanosomatid CaaX protease activities and identifies their potential targets

David Z. Mokry, Surya P. Manandhar, Kristen A. Chicola, George M. Santangelo, and Walter K. Schmidt^*

Department of Biochemistry and Molecular Biology, The University of Georgia, 120 Green Street, Athens, GA 30602; Department of Biological Sciences, The University of Southern Mississippi, 118 College Drive, Hattiesburg, MS 39406

^* To whom correspondence should be addressed. Email: wschmidt{at}bmb.uga.edu.

The CaaX tetrapeptide motif typically directs three sequential post-translational modifications: namely, isoprenylation, proteolysis, and carboxyl methylation. In all eukaryotic systems evaluated to date, two CaaX proteases (Rce1 and Ste24/Afc1) have been identified. While the Trypanosoma brucei genome also encodes two putative CaaX proteases, the lack of detectable Tb Ste24 activity in trypanosome cell extracts has suggested that CaaX proteolytic activity within this organism is solely attributed to Tb Rce1 (20). In this study, we demonstrate that both Tb Rce1 and Tb Ste24 are enzymatically active when heterologously expressed in yeast. Using a-factor and GTPase reporters, we demonstrate that Tb Rce1 and Tb Ste24 possess partially overlapping specificities much like, but not identical to, their fungal and human counterparts. Of interest, a CaaX motif found on a trypanosomal Hsp40 protein was not cleaved by either Tb CaaX protease when examined in the context of the yeast a-factor reporter, but was cleaved by both in the context of the Hsp40 protein itself when evaluated using an in vitro radiolabeling assay. We further demonstrate that Tb Rce1 is sensitive to small molecules previously identified as inhibitors of the yeast and human CaaX proteases, and that a subset of these compounds disrupt Tb Rce1-dependent localization of our GTPase reporter in yeast. Together, our results suggest the conserved presence of two CaaX proteases in trypanosomatids, identify an Hsp40 protein as a substrate of both Tb CaaX proteases, support the potential use of small molecule CaaX protease inhibitors as tools for cell biological studies on the trafficking of CaaX proteins, and provide evidence that protein context influences Tb CaaX protease specificity.