The CaaX tetrapeptide motif typically directs three sequential posttranslational modifications, namely, isoprenylation, proteolysis, and carboxyl methylation. In all eukaryotic systems evaluated to date, two CaaX proteases (Rce1 and Ste24/Afc1) have been identified. Although the Trypanosoma brucei genome also encodes two putative CaaX proteases, the lack of detectable T. brucei Ste24 activity in trypanosome cell extracts has suggested that CaaX proteolytic activity within this organism is solely attributed to T. brucei Rce1 (J. R. Gillespie et al., Mol. Biochem. Parasitol. 153:115-124. 2007). In this study, we demonstrate that both T. brucei Rce1 and T. brucei Ste24 are enzymatically active when heterologously expressed in yeast. Using a-factor and GTPase reporters, we demonstrate that T. brucei Rce1 and T. brucei 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 T. brucei 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 T. brucei 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 T. brucei 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 T. brucei 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 T. brucei CaaX protease specificity.Certain isoprenylated proteins are synthesized as precursors having a highly degenerate C-terminal tetrapeptide CaaX motif (C, cysteine; a, aliphatic amino acid; X, one of several amino acids). This motif typically directs three posttranslational modifications that include covalent attachment of an isoprenoid lipid to the cysteine residue, followed by endoproteolytic removal of the terminal three residues (i.e., aaX), and lastly, carboxyl methyl esterification of the farnesylated cysteine (49,50). Relevant examples of proteins subject to the above modifications, also referred to as CaaX proteins, include the Ras and Ras-related GTPases, G␥ subunits, prelamin A, members of the Hsp40 family of chaperones, and fungal mating pheromones.Isoprenylation of CaaX proteins is performed by either the farnesyltransferase (FTase) or the geranylgeranyl transferase I (GGTase I). The particular isoprenoid attached, C15 farnesyl or C20 geranylgeranyl, respectively, depends in part on the sequence of the CaaX motif (8,26,31). Proteolysis of isoprenylated intermediates is carried out by the otherwise unrelated Rce1p (Ras converting enzyme 1) and Ste24p (steril...