The Ras converting enzyme (RCE) promotes a proteolytic activity that is required for the maturation of Ras, the yeast a-factor mating pheromone, and certain other proteins whose precursors bear a C-terminal CAAX tetrapeptide motif. Despite the physiological importance of RCE, the enzymatic mechanism of this protease remains undefined. In this study, we have evaluated the substrate specificity of RCE orthologs from yeast (Rce1p), worm, plant, and human and have determined the importance of conserved residues toward enzymatic activity.

The budding yeast Saccharomyces cerevisiae provides a unique opportunity for study of the microtubule-based motor proteins that participate in mitotic spindle function. The genome of Saccharomyces encodes a relatively small and genetically tractable set of microtubule-based motor proteins. The single cytoplasmic dynein and five of the six kinesin-related proteins encoded have been implicated in mitotic spindle function. Each motor protein is unique in amino acid sequence. On account of functional overlap, no single motor is uniquely required for cell viability, however. The ability to create and analyze multiple mutants has allowed experimental dissection of the roles performed by each mitotic motor. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle (kinesin-related Cin8p, Kip1p, Kip3p and Kar3p). Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell (dynein and kinesin-related Kip2p, Kip3p and Kar3p). The six motors apparently contribute three fundamental activities to spindle function: motility, microtubule cross-linking and regulation of microtubule dynamics.

Saccharomyces cerevisiae Cin8p belongs to the BimC family of kinesin-related motor proteins that are essential for spindle assembly. Cin8p levels were found to oscillate in the cell cycle due in part to a high rate of degradation imposed from the end of mitosis through the G1 phase. Cin8p degradation required the anaphase-promoting complex ubiquitin ligase and its late mitosis regulator Cdh1p but not the early mitosis regulator Cdc20p. Cin8p lacks a functional destruction box sequence that is found in the majority of anaphase-promoting complex substrates. We carried out an extensive mutagenesis study to define the cis-acting sequence required for Cin8p degradation in vivo. The C terminus of Cin8p contains two elements required for its degradation: 1) a bipartite destruction sequence composed of a KEN-box plus essential residues within the downstream 22 amino acids and 2) a nuclear localization signal. The bipartite destruction sequence appears in other BimC kinesins as well. Expression of nondegradable Cin8p showed very mild phenotypic effects, with an increase in the fraction of mitotic cells with broken spindles.

The Rce1p protease is required for the maturation of the Ras GTPase and certain other isoprenylated proteins and is considered a chemotherapeutic target. To identify new small-molecule inhibitors of Rce1p, the authors screened the National Cancer Institute Diversity Set compound library using in vitro assays to monitor the proteolytic processing of peptides derived from Ras and the yeast a-factor mating pheromone. Of 46 inhibitors initially identified with a Ras-based assay, only 9 were effective in the pheromone-based assay. The IC(50) values of these 9 compounds were in the low micromolar range for both yeast (6-35 microM) and human Rce1p (0.4-46 microM). Four compounds were somewhat Rce1p selective in that they partially inhibited the Ste24p protease and did not inhibit Ste14p isoprenylcysteine carboxyl methyltransferase, 2 enzymes also involved in the maturation of isoprenylated proteins. The remaining 5 compounds inhibited all 3 enzymes. The 2 most Rce1p-selective agents were ineffective trypsin inhibitors, further supporting the specificity of these agents for Rce1p. The 5 least specific compounds formed colloidal aggregates, a proposed common feature of promiscuous inhibitors. Interestingly, the most specific Rce1p inhibitor also formed a colloidal aggregate. In vivo studies revealed that treatment of wild-type yeast with 1 compound induced a Ras2p delocalization phenotype that mimics observed effects in rce1 ste24 null yeast. The 9 compounds identified in this study represent new tools for understanding the enzymology of postisoprenylation-modifying enzymes and provide new insight for the future development of Rce1p inhibitors.

The modifications occurring to CaaX proteins have largely been established using few reporter molecules (e.g. Ras, yeast a-factor mating pheromone). These proteins undergo three coordinated COOH-terminal events: isoprenylation of the cysteine, proteolytic removal of aaX, and COOH-terminal methylation. Here, we investigated the coupling of these modifications in the context of the yeast Ydj1p chaperone. We provide genetic, biochemical, and biophysical evidence that the Ydj1p CaaX motif is isoprenylated but not cleaved and carboxylmethylated. Moreover, we demonstrate that Ydj1p-dependent thermotolerance and Ydj1p localization are perturbed when alternative CaaX motifs are transplanted onto Ydj1p. The abnormal phenotypes revert to normal when post-isoprenylation events are genetically interrupted. Our findings indicate that proper Ydj1p function requires an isoprenylatable CaaX motif that is resistant to post-isoprenylation events. These results expand on the complexity of protein isoprenylation and highlight the impact of post-isoprenylation events in regulating the function of Ydj1p and perhaps other CaaX proteins.DOI: http://dx.doi.org/10.7554/eLife.15899.001

The CaaX proteases Rce1p and Ste24p can independently promote a proteolytic step required for the maturation of certain isoprenylated proteins. Although functionally related, Rce1p and Ste24p are unrelated in primary sequence. They have distinct enzymatic properties, which are reflected in part by their distinct inhibitor profiles. Moreover, Rce1p has an undefined catalytic mechanism, whereas Ste24p is an established zinc-dependent metalloprotease. This study demonstrates that both enzymes are inhibited by peptidyl (acyloxy)methyl ketones (AOMKs), making these compounds the first documented dual specificity inhibitors of the CaaX proteases. Further investigation of AOMK-mediated inhibition reveals that varying the peptidyl moiety can significantly alter the inhibitory properties of AOMKs toward Rce1p and Ste24p and that these enzymes display subtle differences in sensitivity to AOMKs. This observation suggests that this compound class could potentially be engineered to be selective for either of the CaaX proteases. We also demonstrate that the reported sensitivity of Rce1p to TPCK is substrate-dependent, which significantly alters the interpretation of certain reports having used TPCK sensitivity for mechanistic classification of Rce1p. Finally, we show that an AOMK inhibits the isoprenylcysteine carboxyl methyltransferase Ste14p. In sum, our observations raise important considerations regarding the specificity of agents targeting enzymes involved in the maturation of isoprenylated proteins, some of which are being developed as anti-cancer therapeutic agents.

Kinesin-5 motor proteins are evolutionarily conserved and perform essential roles in mitotic spindle assembly and spindle elongation during anaphase. Previous studies demonstrated a specialized homotetrameric structure with two pairs of catalytic domains, one at each end of a dumbbell-shaped molecule. This suggests that they perform their spindle roles by cross-linking and sliding antiparallel spindle microtubules. However, the exact kinesin-5 sequence elements that are important for formation of the tetrameric complexes have not yet been identified. In addition, it has not been demonstrated that the homotetrameric form of these proteins is essential for their biological functions. Thus, we investigated a series of Saccharomyces cerevisiae Cin8p truncations and internal deletions, in order to identify structural elements in the Cin8p sequence that are required for Cin8p functionality, spindle localization, and multimerization. We found that all variants of Cin8p that are functional in vivo form tetrameric complexes. The first coiled-coil domain in the stalk of Cin8p, a feature that is shared by all kinesin-5 homologues, is required for its dimerization, and sequences in the last part of the stalk, specifically those likely involved in coiled-coil formation, are required for Cin8p tetramerization. We also found that dimeric forms of Cin8p that are nonfunctional in vivo can nonetheless bind to microtubules. These findings suggest that binding of microtubules is not sufficient for the functionality of Cin8p and that microtubule cross-linking by the tetrameric complex is essential for Cin8p mitotic functions.Mitotic chromosome segregation is the mechanism by which duplicated genomic information is transmitted to daughter cells during cell division. This essential process is mediated by the mitotic spindle, a highly dynamic, microtubule-based structure that undergoes a distinct set of morphological changes. Many of these changes are achieved by the action of molecular motors from the kinesin-5 (BimC) family, which use ATP hydrolysis to unidirectionally move along microtubules. Members of the kinesin-5 family are conserved in the amino acid sequence of the motor (forceproducing) domain and apparently perform similar roles in many different cell types (1-7). Kinesin-5 motors are required for bipolar spindle assembly, and elimination of their function blocks this essential early mitotic step in fungal, insect, and mammalian cells (8 -11). The yeast Saccharomyces cerevisiae expresses two kinesin-5 motors that overlap in function, Cin8p and Kip1p. Although neither is individually essential, one of the pair is required for viability (2, 3, 12). Loss of KIP1 function causes less severe phenotypes than loss of CIN8, suggesting that Cin8p is more important for successful yeast spindle function (2). Aside from their essential role in spindle assembly, Cin8p and Kip1p are also required for the maintenance of spindle bipolarity following assembly and are responsible for producing most of the spindle-elongating force during anaph...

In vitro and in silico studies of the CaaX-type prenyl transferases suggest a wider array of prenylatable sequences than those determined in vivo. Berger and Kim et al. investigate whether this disconnect is due to use of...