The CaaX Proteases, Afc1p and Rce1p, Have Overlapping but Distinct Substrate Specificities

Trueblood, C E; Boyartchuk, Victor L.; Picologlou, Elizabeth A.; Rozema, David B.; Poulter, C. Dale; Rine, Jasper

doi:10.1128/mcb.20.12.4381-4392.2000

Cited by 94 publications

(133 citation statements)

References 49 publications

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“…In Saccharomyces cerevisiae, STE24 (AFC1) and RCE1 act redundantly in processing the a-factor. Only RCE1, however, is responsible for processing RAS2, whereas the only known substrate of STE24 (AFC1) is the a-factor (9,17). Fibroblasts isolated from rce1 Ϫ/Ϫ knockout mice were unable to process Ras proteins (18), whereas fibroblasts isolated from zmpste24 Ϫ/Ϫ knockout mice were able to process both Ras and other proteins (19).…”

mentioning

confidence: 88%

“…Growth Arrest Pheromone Diffusion (Halo) Assays-The assays were carried out essentially as described previously (17) with the following modifications. Three l of MATa cell slurry (ϳ10 7 cells) were spotted onto a solid, rich medium (YPD) plate containing 0.01% Triton X-100 that had been spread with a lawn (ϳ2 ϫ 10 6 cells) of the MAT␣ sst2 cells (JRY3443).…”

Section: Methodsmentioning

confidence: 99%

“…a-Factor produces a growth inhibition zone known as a halo when applied to a mat of sst2 MATa cells, because of the growth arrest of the latter at the G 1 phase of the cell cycle. The size of the halo corresponds to the concentration of a-factor and hence to the amount of active pheromone produced by the MATa cells (17). For it to be exported and functional, the 36-amino acid a-factor precursor undergoes farnesylation proteolytic trimming of the AAX (VIA) moiety by either STE24 or RCE1 carboxymethylation and two N-terminal proteolytic cleavage events, the first of which is catalyzed by STE24 (Fig.…”

Section: Atste24 Is Homologous To Yeast and Human Proteins-mentioning

confidence: 99%

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The Arabidopsis AtSTE24 Is a CAAXProtease with Broad Substrate Specificity

Bracha

Lavy

Yalovsky

2002

Journal of Biological Chemistry

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Following prenylation, the proteins are subject to two prenyl-dependent modifications at their C-terminal end, which are required for their subcellular targeting. First, the three C-terminal residues of the CAAX box prenylation signaling motif are removed, which is followed by methylation of the free carboxyl group of the prenyl cysteine moiety. An Arabidopsis homologue of the yeast CAAX protease STE24 (AFC1) was cloned and expressed in rce1⌬ ste24⌬ mutant yeast to demonstrate functional complementation. The petunia calmodulin CaM53 is a prenylated protein terminating in a CTIL CAAX box. Coupled methylation proteolysis assays demonstrated the processing of CaM53 by AtSTE24. In addition, AtSTE24 promoted plasma membrane association of the GFP-RacU88402 fusion protein, which terminates with a CLLM CAAX box. Interestingly, a plant homologue of the second and major CAAX protease in yeast and animal cells, RCE1, was not identified despite the availability of vast amounts of sequence data. Taken together, these data suggest that AtSTE24 may process several prenylated proteins in plant cells, unlike its yeast homologue, which processes only a-mating factor, and its mammalian homologue, for which prenyl-CAAX substrates have not been established. Transient expression of GFPAtSTE24 in leaf epidermal cells of Nicotiana benthamiana showed that AtSTE24 is exclusively localized in the endoplasmic reticulum, suggesting that prenylated proteins in plants are first targeted to the endoplasmic reticulum following their prenylation.

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confidence: 88%

Section: Methodsmentioning

confidence: 99%

Section: Atste24 Is Homologous To Yeast and Human Proteins-mentioning

confidence: 99%

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The Arabidopsis AtSTE24 Is a CAAXProtease with Broad Substrate Specificity

Bracha

Lavy

Yalovsky

2002

Journal of Biological Chemistry

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“…Although functionally redundant for the endoproteolysis of the yeast a-factor CaaX motif, Rce1p and Ste24p have distinct, albeit overlapping, substrate specificities when tested in vivo against a panel of CaaX motifs 23 . Notably, the processing of Ras and most other CaaX proteins is solely dependent on Rce1p; a-factor is the only known physiological exception, though more may exist.…”

Section: Enzymatic Processing Of Prenylated Proteinsmentioning

confidence: 99%

Therapeutic intervention based on protein prenylation and associated modifications

et al. 2006

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In eukaryotic cells, a specific set of proteins are modified by C-terminal attachment of 15-carbon farnesyl groups or 20-carbon geranylgeranyl groups that function both as anchors for fixing proteins to membranes and as molecular handles for facilitating binding of these lipidated proteins to other proteins. Additional modification of these prenylated proteins includes C-terminal proteolysis and methylation, and attachment of a 16-carbon palmitoyl group; these modifications augment membrane anchoring and alter the dynamics of movement of proteins between different cellular membrane compartments. The enzymes in the protein prenylation pathway have been isolated and characterized. Blocking protein prenylation is proving to be therapeutically useful for the treatment of certain cancers, infection by protozoan parasites and the rare genetic disease Hutchinson-Gilford progeria syndrome.Isoprenoids are lipids made of five-carbon blocks, and they constitute one of the main classes of natural products. A subset of isoprenoids called prenyl groups are found on a variety of biological substances, including proteins. Prenylated proteins and peptides are found in most (if not all) eukaryotes. They arise from the post-translational attachment of 15-carbon farnesyl or 20-carbon geranylgeranyl groups to the C-terminal segment of proteins. Protein prenyl groups not only are hydrophobic elements that bind proteins to membranes, but, at least in some cases, they also function as molecular handles that bind to hydrophobic grooves on the surface of soluble protein factors; these factors then remove the prenylated protein from the membrane in a regulated manner. NIH Public Access Author ManuscriptNat Chem Biol. Author manuscript; available in PMC 2010 June 28. Published in final edited form as:Nat Chem Biol. 2006 October ; 2(10): 518-528. doi:10.1038/nchembio818. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptProtein prenylation has been vigorously studied over the past ~15 years because it is found on several signaling proteins (including heterotrimeric G proteins) that connect cell surface receptors to intracellular effectors, and also on Ras proteins, one of the most common oncoproteins found in human tumors. Ras proteins serve as molecular switches at cellular membranes to control propagation of growth signals from cell surface receptors to nuclear transcription factors. Because Ras mutations are important in many human cancers, there has been extensive focus on Ras prenylation. Discovery of protein prenyl groups and structural varietiesThe first reports of prenylated proteins and peptides described the secreted pheromone peptides from jelly fungi 1,2 , whose structure resembles that of the well-known a-factor mating pheromone from baker's yeast (Saccharomyces cerevisiae), which contains a cysteine methylester farnesylated at the C terminus 3 . In the 1980s, studies on the timing of cholesterol biosynthesis with respect to the cell cycle in human cells led to the discovery that a compound deriv...

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“…Haploid MATa yeast lacking both RCE1 and STE24 (ste24⌬rce1⌬) grew normally but were unable to produce mature a-factor and therefore were sterile (5). Interestingly, Rce1p and Ste24p exhibited subtle differences in substrate specificities (5)(6)(7). Both proteins were capable of cleaving the carboxyl terminus of wild-type a-factor, which terminates in CVIA.…”

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Biochemical Studies of Zmpste24-deficient Mice

Leung¹,

Schmidt²,

Bergö³

et al. 2001

Journal of Biological Chemistry

100

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Genetic studies in Saccharomyces cerevisiae identified two genes, STE24 and RCE1, involved in cleaving the three carboxyl-terminal amino acids from isoprenylated proteins that terminate with a CAAX sequence motif. Ste24p cleaves the carboxyl-terminal "-AAX" from the yeast mating pheromone a-factor, whereas Rce1p cleaves the -AAX from both a-factor and Ras2p. Ste24p also cleaves the amino terminus of a-factor. The mouse genome contains orthologues for both yeast RCE1 and STE24. We previously demonstrated, with a gene-knockout experiment, that mouse Rce1 is essential for development and that Rce1 is entirely responsible for the carboxyl-terminal proteolytic processing of the mouse Ras proteins. In this study, we cloned mouse Zmpste24, the orthologue for yeast STE24 and showed that it could promote a-factor production when expressed in yeast. Then, to assess the importance of Zmpste24 in development, we generated Zmpste24-deficient mice. Unlike the Rce1 knockout mice, Zmpste24-deficient mice survived development and were fertile. Since no natural substrates for mammalian Zmpste24 have been identified, yeast a-factor was used as a surrogate substrate to investigate the biochemical activities in membranes from the cells and tissues of Zmpste24-deficient mice. We demonstrate that Zmpste24-deficient mouse membranes, like Ste24p-deficient yeast membranes, have diminished CAAX proteolytic activity and lack the ability to cleave the amino terminus of the a-factor precursor. Thus, both enzymatic activities of yeast Ste24p are conserved in mouse Zmpste24, but these enzymatic activities are not essential for mouse development or for fertility.Proteins that terminate in a carboxyl-terminal CAAX motif 1 undergo three sequential enzymatic processing events, farnesylation or geranylgeranylation of the cysteine, endoproteolytic release of the last three amino acid residues of the protein (i.e. removal of the -AAX), and methylation of the new carboxyl terminus of the protein by isoprenylcysteine carboxyl methyltransferase (1, 2). The yeast genes responsible for the farnesylation and methylation steps were identified more than a decade ago (3, 4), but the identification of the genes responsible for the middle processing step, the endoprotease step, remained elusive for years (2). Ultimately, however, Boyartchuk and co-workers (5) applied a novel genetic selection scheme and identified two yeast genes, RCE1 and STE24 (AFC1), involved in the carboxyl-terminal endoproteolytic processing of isoprenylated CAAX proteins. Rce1p is a protease involved in the carboxyl-terminal processing of both a-factor and the yeast Ras protein, Ras2p. Ste24p (Afc1p), a zinc metalloprotease, lacked activity against Ras2p but did process a-factor. Haploid MATa yeast lacking both RCE1 and STE24 (ste24⌬rce1⌬) grew normally but were unable to produce mature a-factor and therefore were sterile (5). Interestingly, Rce1p and Ste24p exhibited subtle differences in substrate specificities (5-7). Both proteins were capable of cleaving the carboxyl terminus of wild-...

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The CaaX Proteases, Afc1p and Rce1p, Have Overlapping but Distinct Substrate Specificities

Cited by 94 publications

References 49 publications

The Arabidopsis AtSTE24 Is a CAAXProtease with Broad Substrate Specificity

The Arabidopsis AtSTE24 Is a CAAXProtease with Broad Substrate Specificity

Therapeutic intervention based on protein prenylation and associated modifications

Biochemical Studies of Zmpste24-deficient Mice

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