Topology of the Yeast Ras Converting Enzyme As Inferred from Cysteine Accessibility Studies

The Ras converting enzyme (Rce1p) is required for the proteolytic maturation of a large subset of isoprenylated proteins (1, 2). Relevant biomedical targets of Rce1p include the Ras GTPases. These proteins and other targets are typically referred to as CaaX proteins due to the presence of a COOH-terminal tetrapeptide sequence (cysteine, aliphatic, aliphatic, one of several amino acids) that serves as a recognition motif for isoprenylation. Rce1p is responsible for removal of the aaX portion of the motif subsequent to isoprenylation. Proteolysis is followed by carboxylmethylation of the COOH-terminal isoprenylated cysteine. In some instances, CaaX proteins undergo additional modification involving S-acylation (e.g. N and H-Ras) or proteolysis (e.g. prelamin A and yeast a-factor precursor). The above post-translational modifications modulate the membrane partitioning and protein-protein interaction properties of CaaX proteins, and interfering with these steps is considered having anti-cancer therapeutic value. Rce1p is a membrane protein that is localized to the endoplasmic reticulum (ER) membrane (3, 4). It has not yet been purified in active form, and its three-dimensional structure remains undetermined. Hydropathy analyses predict it to be an integral membrane protein with multiple membrane spanning segments (i.e. polytopic), ranging from 6-10 spans depending on the algorithm used (5) (Table S2). A polytopic protease is unusual but not without precedent. Notable examples include Ste24p with which Rce1p has partially overlapping activity, the presenilins that cleave the precursor of the Aβ peptide and Notch, the signal peptide peptidase (SPP) and related SPP-family members, the Site 2 protease (S2P) that is involved in the proteolytic liberation of membrane anchored transcription factors, and Rhomboid that regulates a range of activities, including production of a cell signaling factor, parasite invasion, mitochondrial homeostasis, and potentially bacterial quorum sensing (6, 7). Although hydropathy analysis consistently identifies Rce1p as being a membrane protein, it is important to note that hydropathy algorithms only identify hydrophobic stretches within proteins. These algorithms do not distinguish between bitopic (fully transmembrane) and monotopic orientation (a hydrophobic loop that dips into and out of one leaflet of the bilayer; also called a re-entrant helix). The results of this study suggest that Rce1p likely has both types of hydrophobic segments. The catalytic mechanism of Rce1p has also eluded classification, with cysteine and metalloprotease mechanisms being most often cited and a novel mechanism even being possible (5, 8, 9). Three residues (a glutamate and two histidines) have been identified that are conserved among Rce1p orthologs, including those from prokaryotic species (10, 11). These residues are required for the activity of yeast and trypanosomal Rce1p, suggesting that they are part of the Rce1p active-site (5, 12). As part of an active-site, these residues are hypothesized to lie within the interfacial region between the ER membrane and cytosol where interaction with isoprenylated proteins would be expected. To test this hypothesis, we performed a careful topological study of yeast Rce1p, primarily using a membrane impermeant PEGylated sulfhydryl modifying agent (i.e. PEG-Maleimide 5000; PEG-Mal) to probe the accessibility of naturally occurring or introduced cysteine residues (13, 14). We complement these studies with an assessment of the glycosylation potential of the NH2 and COOH terminal ends of Rce1p, and also provide hydropathy analyses of Rce1p and related orthologs. The inferred topology localizes the essential residues to the cytoplasmic side of the membrane bilayer, as expected.