title: Mechanism of farnesylated CAAX protein processing by the intramembrane protease Rce1.
abstract: CAAX proteins have essential roles in multiple signalling pathways, controlling processes such as proliferation, differentiation and carcinogenesis. The ∼120 mammalian CAAX proteins function at cellular membranes and include the Ras superfamily of small GTPases, nuclear lamins, the γ-subunit of heterotrimeric GTPases, and several protein kinases and phosphatases. The proper localization of CAAX proteins to cell membranes is orchestrated by a series of post-translational modifications of the carboxy-terminal CAAX motifs (where C is cysteine, A is an aliphatic amino acid and X is any amino acid). These reactions involve prenylation of the cysteine residue, cleavage at the AAX tripeptide and methylation of the carboxyl-prenylated cysteine residue. The major CAAX protease activity is mediated by Rce1 (Ras and a-factor converting enzyme 1), an intramembrane protease (IMP) of the endoplasmic reticulum. Information on the architecture and proteolytic mechanism of Rce1 has been lacking. Here we report the crystal structure of a Methanococcus maripaludis homologue of Rce1, whose endopeptidase specificity for farnesylated peptides mimics that of eukaryotic Rce1. Its structure, comprising eight transmembrane α-helices, and catalytic site are distinct from those of other IMPs. The catalytic residues are located ∼10 Å into the membrane and are exposed to the cytoplasm and membrane through a conical cavity that accommodates the prenylated CAAX substrate. We propose that the farnesyl lipid binds to a site at the opening of two transmembrane α-helices, which results in the scissile bond being positioned adjacent to a glutamate-activated nucleophilic water molecule. This study suggests that Rce1 is the founding member of a novel IMP family, the glutamate IMPs.
authors.affiliation: Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Manolaridis
authors.affiliation: Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
authors.forename: Kiran
authors.initials: K
authors.lastname: Kulkarni
authors.affiliation: Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
authors.forename: Roger B
authors.initials: RB
authors.lastname: Dodd
authors.affiliation: Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.|JST, Research Acceleration Program, Membrane Protein, Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ogasawara
authors.affiliation: Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
authors.forename: Ziguo
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3LY, UK.
authors.forename: Ganka
authors.initials: G
authors.lastname: Bineva
authors.affiliation: Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London, WC2A 3LY, UK.
authors.forename: Nicola O'
authors.initials: NO
authors.lastname: Reilly
authors.affiliation: Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
authors.forename: Sarah J
authors.initials: SJ
authors.lastname: Hanrahan
authors.affiliation: Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thompson
authors.affiliation: Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
authors.forename: Nora
authors.initials: N
authors.lastname: Cronin
authors.affiliation: Department of Cell Biology, Graduate School of Medicine, Kyoto University, Yoshida-konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.|JST, Research Acceleration Program, Membrane Protein, Crystallography Project, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, 606-8501, Japan.|Division of Molecular Biosciences, Imperial College, London, SW7 2AZ, UK.
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.affiliation: Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK.
authors.forename: David
authors.initials: D
authors.lastname: Barford
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism; D017017:Methanococcus / Q000201:enzymology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000145:classification / Q000378:metabolism*; D054876:Prenylation; D017434:Protein Structure, Tertiary; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction; D013379:Substrate Specificity
chemical_list: D019843:Archaeal Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C102898:GlpG protein, E coli; D008565:Membrane Proteins; D018698:Glutamic Acid; D010450:Endopeptidases; D010447:Peptide Hydrolases; C491326:RCE1 protein, human; D008666:Metalloendopeptidases; C120130:ZMPSTE24 protein, human; D016283:Proto-Oncogene Proteins p21(ras); D003545:Cysteine