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PubMed1996

Molecular studies of CtpA, the carboxyl-terminal processing protease for the D1 protein of the photosystem II reaction center in higher plants.

The D1 reaction center protein of the Photosystem II complex in green plants is synthesized with a short carboxyl-terminal extension. Proteolytic cleavage and removal of this extension peptide in the thylakoid lumen are necessary for the assembly of a manganese cluster that is essential for the oxygen evolution activity of Photosystem II. We have isolated cDNAs encoding CtpA, the carboxyl-terminal processing protease for the D1 protein, from two higher plants, spinach and barley. In each of these organisms, CtpA is encoded by a single copy nuclear gene, and its steady-state mRNA levels are light-regulated. The CtpA protein is detectable in etiolated material, and its level increases approximately 5-fold upon illumination. Moreover, the CtpA gene is expressed in shoot tissues and not in roots. In its precursor form, the CtpA protein harbors a bipartite transit sequence characteristic for thylakoid lumenal proteins. Cell fractionation studies demonstrated that CtpA is associated with thylakoid membranes and is resistant to treatments with thermolysin, consistent with its localization in the lumen of thylakoids. Comparisons of the sequence of the higher plant CtpA enzyme with those of other related carboxyl-terminal processing proteases suggest that these proteins constitute a new family of proteases.

Algal Proteins↗
PubMed1997

Endoprotease activities other than furin and PACE4 with a role in processing of HIV-I gp160 glycoproteins in CHO-K1 cells.

We addressed the question of whether furin is the endoprotease primarily responsible for processing the human immunodeficiency virus type I (HIV-I) envelope protein gp160 in mammalian cells. The furin-deficient Chinese hamster ovary (CHO)-K1 strain RPE.40 processed gp160 as efficiently as wild-type CHO-K1 cells in vivo. Although furin can process gp160 in vitro, this processing is probably not physiologically relevent, because it occurs with very low efficiency. PACE4, a furin homologue, allowed processing of gp160 when both were expressed in RPE.40 cells. Further, PACE4 participated in the activation of a calcium-independent protease activity in RPE.40 cells, which efficiently processed the gp160 precursor in vitro. This calcium-independent protease activity was not found in another furin-deficient cell strain, 7.P15, selected from the monkey kidney cell line COS-7.

Adenovirus E1A Proteins↗
PubMed1998

Golgi localization and functionally important domains in the NH2 and COOH terminus of the yeast CLC putative chloride channel Gef1p.

GEF1 encodes the single CLC putative chloride channel in yeast. Its disruption leads to a defect in iron metabolism (Greene, J. R., Brown, N. H., DiDomenico, B. J., Kaplan, J., and Eide, D. (1993) Mol. Gen. Genet. 241, 542-553). Since disruption of GEF2, a subunit of the vacuolar H+-ATPase, leads to a similar phenotype, it was previously suggested that the chloride conductance provided by Gef1p is necessary for vacuolar acidification. We now show that gef1 cells indeed grow less well at less acidic pH. However, no defect in vacuolar acidification is apparent from quinacrine staining, and Gef1p co-localizes with Mnt1p in the medial Golgi. Thus, Gef1p may be important in determining Golgi pH. Systematic alanine scanning of the amino and the carboxyl terminus revealed several regions essential for Gef1p localization and function. One sequence (FVTID) in the amino terminus conforms to a class of sorting signals containing aromatic amino acids. This was further supported by point mutations. Alanine scanning of the carboxyl terminus identified a stretch of roughly 25 amino acids which coincides with the second CBS domain, a conserved protein motif recently identified. Mutations in the first CBS domain also destroyed proper function and localization. The second CBS domain can be transplanted to the amino terminus without loss of function, but could not be replaced by the corresponding domain of the homologous mammalian channel ClC-2.

Amino Acid Sequence↗
PubMed1999

Cloning and characterization of a mammalian prenyl protein-specific protease.

Proteins containing C-terminal "CAAX" sequence motifs undergo three sequential post-translational processing steps: modification of the cysteine with either a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenyl lipid, proteolysis of the C-terminal -AAX tripeptide, and methylation of the carboxyl group of the now C-terminal prenylcysteine. A putative prenyl protein protease in yeast, designated Rce1p, was recently identified. In this study, a portion of a putative human homologue of RCE1 (hRCE1) was identified in a human expressed sequence tag data base, and the corresponding cDNA was cloned. Expression of hRCE1 was detected in all tissues examined. Both yeast and human RCE1 proteins were produced in Sf9 insect cells by infection with a recombinant baculovirus; membrane preparations derived from the infected Sf9 cells exhibited a high level of prenyl protease activity. Recombinant hRCE1 so produced recognized both farnesylated and geranylgeranylated proteins as substrates, including farnesyl-Ki-Ras, farnesyl-N-Ras, farnesyl-Ha-Ras, and the farnesylated heterotrimeric G protein Ggamma1 subunit, as well as geranylgeranyl-Ki-Ras and geranylgeranyl-Rap1b. The protease activity of hRCE1 activity was specific for prenylated proteins, because unprenylated peptides did not compete for enzyme activity. hRCE1 activity was also exquisitely sensitive to a prenyl peptide analogue that had been previously described as a potent inhibitor of the prenyl protease activity in mammalian tissues. These data indicate that both the yeast and the human RCE1 gene products are bona fide prenyl protein proteases and suggest that they play a major role in the processing of CAAX-type prenylated proteins.

Amino Acid Sequence↗
PubMed1999

Disruption of the mouse Rce1 gene results in defective Ras processing and mislocalization of Ras within cells.

Little is known about the enzyme(s) required for the endoproteolytic processing of mammalian Ras proteins. We identified a mouse gene (designated Rce1) that shares sequence homology with a yeast gene (RCE1) implicated in the proteolytic processing of Ras2p. To define the role of Rce1 in mammalian Ras processing, we generated and analyzed Rce1-deficient mice. Rce1 deficiency was lethal late in embryonic development (after embryonic day 15.5). Multiple lines of evidence revealed that Rce1-deficient embryos and cells lacked the ability to endoproteolytically process Ras proteins. First, Ras proteins from Rce1-deficient cells migrated more slowly on SDS-polyacrylamide gels than Ras proteins from wild-type embryos and fibroblasts. Second, metabolic labeling of Rce1-deficient cells revealed that the Ras proteins were not carboxymethylated. Finally, membranes from Rce1-deficient fibroblasts lacked the capacity to proteolytically process farnesylated Ha-Ras, N-Ras, and Ki-Ras or geranylgeranylated Ki-Ras. The processing of two other prenylated proteins, the farnesylated Ggamma1 subunit of transducin and geranylgeranylated Rap1B, was also blocked. The absence of endoproteolytic processing and carboxymethylation caused Ras proteins to be mislocalized within cells. These studies indicate that Rce1 is responsible for the endoproteolytic processing of the Ras proteins in mammals and suggest a broad role for this gene in processing other prenylated CAAX proteins.

Animals↗
PubMed1999

Autocatalytic processing of site-1 protease removes propeptide and permits cleavage of sterol regulatory element-binding proteins.

Site-1 protease (S1P) is a subtilisin-related protease that cleaves sterol regulatory element-binding proteins (SREBPs) in the endoplasmic reticulum lumen, thereby initiating a process by which the transcriptionally active NH(2)-terminal fragments of SREBPs are released from membranes. In the current experiments, we transfected cDNAs encoding epitope-tagged hamster S1P into HEK-293 cells or mutant hamster cells that lack S1P. Protease protection assays showed that the bulk of S1P is in the endoplasmic reticulum lumen, anchored by a COOH-terminal membrane-spanning segment. Cleavage of the NH(2)-terminal signal sequence of S1P generates S1P-A (amino acids 23-1052), which is inactive. The protein is self-activated by an intramolecular cleavage at Site-B, generating S1P-B (amino acids 138-1052) and liberating a 115-amino acid propeptide that is secreted intact into the medium. The sequence at Site-B is RSLK, which differs from the RSVL sequence at the cleavage site in SREBP-2. S1P-B is further cleaved at an internal RRLL sequence to yield S1P-C (amino acids 187-1052). Mutational analysis suggests that S1P-B and S1P-C are both active in cleaving SREBP-2 in a fashion that requires SREBP cleavage-activating protein. The activity of S1P-C may be short-lived because it appears to be transported to the Golgi, a site at which SREBP-2 cleavage may not normally occur. These data provide the initial description of the processing of a subtilisin-related protease that controls the level of cholesterol in blood and cells. In an accompanying paper (Cheng, D., Espenshade, P. J., Slaughter, C. A., Jaen, J. C., Brown, M. S., and Goldstein, J. L. (1999), J. Biol. Chem., 274, 22805-22812), we develop an in vitro assay to characterize the activity of purified recombinant S1P.

Amino Acid Sequence↗
PubMed1999

Quantitative characterization of furin specificity. Energetics of substrate discrimination using an internally consistent set of hexapeptidyl methylcoumarinamides.

Furin, an essential mammalian proprotein processing enzyme of the kexin/furin family of subtilisin-related eukaryotic processing proteases, is implicated in maturation of substrates involved in development, signaling, coagulation, and pathogenesis. We examined the energetics of furin specificity using a series of peptidyl methylcoumarinamide substrates. In contrast to previous reports, we found that furin can cleave such substrates with kinetics comparable to those observed with extended peptides and physiological substrates. With the best of these hexapeptidyl methylcoumarinamides, furin displayed k(cat)/K(m) values greater than 10(6) M(-1) s(-1). Furin exhibited striking substrate inhibition with hexapeptide but not tetrapeptide substrates, an observation of significance to the evaluation of peptide-based furin inhibitors. Quantitative comparison of furin and Kex2 recognition at P(1), P(2), and P(4) demonstrates that whereas interactions at P(1) make comparable contributions to catalysis by the two enzymes, furin exhibited a approximately 10-fold lesser dependence on P(2) recognition but a 10-100-fold greater dependence on P(4) recognition. Furin has recently been shown to exhibit P(6) recognition and we found that this interaction contributes approximately 1.4 kcal/mol toward catalysis independent of the nature of the P(4) residue. We have also shown that favorable residues at P(2) and P(6) will compensate for less than optimal residues at either P(1) or P(4). The quantitative analysis of furin and Kex2 specificity sharply distinguish the nature of substrate recognition by the processing and degradative members of subtilisin-related proteases.

Amides↗
PubMed1999

Purification and characterization of the yeast glycosylphosphatidylinositol-anchored, monobasic-specific aspartyl protease yapsin 2 (Mkc7p).

The Saccharomyces cerevisiae YPS2 (formerly MKC7) gene product is a glycosylphosphatidylinositol-linked aspartyl protease that functions as a yeast secretase. Here, the glycosylphosphatidylinositol-linked form of yapsin 2 (Mkc7p) was purified to homogeneity from the membrane fraction of an overexpressing yeast strain. Purified yapsin 2 migrated diffusely in SDS-polyacrylamide gel electrophoresis (molecular mass approximately 200 kDa), suggesting extensive, heterogeneous glycosylation. Studies using internally quenched fluorogenic peptide substrates revealed cleavage by the enzyme carboxyl to Lys or Arg. No cleavage was seen when both Lys and Arg were absent. No significant enhancement was seen with multiple basic residues. However, cleavage always occurred carboxyl to the most COOH-terminal basic residue. V(max)/K(m) was insensitive to P(2) and P(3) residues except that Pro at P(2) blocked cleavage entirely. These results suggest that yapsin 2 is a monobasic amino acid-specific protease that requires a basic residue at P(1) and excludes basic residues from P(1)'. The pH dependence of V(max)/K(m) for a substrate containing a pro-alpha factor cleavage site was bell-shaped, with a maximum near pH 4.0. However, V(max)/K(m) for a substrate mimicking the alpha-secretase site in human beta amyloid precursor protein was optimal near pH 6.0, consistent with cleavage of beta amyloid precursor protein by yapsin 2 when expressed in yeast.

Amino Acid Sequence↗
PubMed2000

Biosynthesis and enzymatic characterization of human SKI-1/S1P and the processing of its inhibitory prosegment.

Biochemical and enzymatic characterization of the novel human subtilase hSKI-1 was carried out in various cell lines. Within the endoplasmic reticulum of LoVo cells, proSKI-1 is converted to SKI-1 by processing of its prosegment into 26-, 24-, 14-, 10-, and 8-kDa products, some of which remain tightly associated with the enzyme. N-terminal sequencing and mass spectrometric analysis were used to map the cleavage sites of the most abundant fragments, which were confirmed by synthetic peptide processing. To characterize its in vitro enzymatic properties, we generated a secreted form of SKI-1. Our data demonstrate that SKI-1 is a Ca(2+)-dependent proteinase exhibiting optimal cleavage at pH 6.5. We present evidence that SKI-1 processes peptides mimicking the cleavage sites of the SKI-1 prosegment, pro-brain-derived neurotrophic factor, and the sterol regulatory element-binding protein SREBP-2. Among the candidate peptides encompassing sections of the SKI-1 prosegment, the RSLK(137)- and RRLL(186)-containing peptides were best cleaved by this enzyme. Mutagenesis of the latter peptide allowed us to develop an efficiently processed SKI-1 substrate and to assess the importance of several P and P' residues. Finally, we demonstrate that, in vitro, recombinant prosegments of SKI-1 inhibit its activity with apparent inhibitor constants of 100-200 nM.

Amino Acid Sequence↗
PubMed2000

Altered distribution of the yeast plasma membrane H+-ATPase as a feature of vacuolar H+-ATPase null mutants.

The effect of vacuolar H(+)-ATPase (V-ATPase) null mutations on the targeting of the plasma membrane H(+)-ATPase (Pma1p) through the secretory pathway was analyzed. Gas1p, which is another plasma membrane component, was used as a control for the experiments with Pma1p. Contrary to Gas1p, which is not affected by the deletion of the V-ATPase complex in the V-ATPase null mutants, the amount of Pma1p in the plasma membrane is markedly reduced, and there is a large accumulation of the protein in the endoplasmic reticulum. Kex2p and Gef1p, which are considered to reside in the post-Golgi vesicles, were suggested as required for the V-ATPase function; hence, their null mutant phenotype should have been similar to the V-ATPase null mutants. We show that, in addition to the known differences between those yeast phenotypes, deletions of KEX2 or GEF1 in yeast do not affect the distribution of Pma1p as the V-ATPase null mutant does. The possible location of the vital site of acidification by V-ATPase along the secretory pathway is discussed.

Cell Membrane↗
PubMed2001

Amino acid residues that are critical for in vivo catalytic activity of CtpA, the carboxyl-terminal processing protease for the D1 protein of photosystem II.

CtpA, a carboxyl-terminal processing protease, is a member of a novel family of endoproteases that includes a tail-specific protease from Escherichia coli. In oxygenic photosynthetic organisms, CtpA catalyzes C-terminal processing of the D1 protein of photosystem II, an essential event for the assembly of a manganese cluster and consequent light-mediated water oxidation. We introduced site-specific mutations at 14 conserved residues of CtpA in the cyanobacterium Synechocystis sp. PCC 6803 to examine their functional roles. Analysis of the photoautotrophic growth capabilities of these mutants, their ability to process precursor D1 protein and hence evolve oxygen, along with an estimation of the protease content in the mutants revealed that five of these residues are critical for in vivo activity of CtpA. Recent x-ray crystal structure analysis of CtpA from the eukaryotic alga Scenedesmus obliquus (Liao, D.-I., Qian, J., Chisholm, D. A., Jordan, D. B. and Diner, B. A. (2000) Nat. Struct. Biol. 7, 749-753) has shown that the residues equivalent to Ser-313 and Lys-338, two of the five residues mentioned above, form the catalytic center of this enzyme. Our in vivo analysis demonstrates that the three other residues, Asp-253, Arg-255, and Glu-316, are also important determinants of the catalytic activity of CtpA.

Algal Proteins↗
PubMed2001

Biochemical studies of Zmpste24-deficient mice.

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.

Amino Acid Sequence↗
PubMed2001

Differential utilization of enzyme-substrate interactions for acylation but not deacylation during the catalytic cycle of Kex2 protease.

Kex2 protease from Saccharomyces cerevisiae is the prototype for a family of eukaryotic proprotein processing proteases belonging to the subtilase superfamily of serine proteases. Kex2 can be distinguished from degradative subtilisins on the basis of stringent substrate specificity and distinct pre-steady-state behavior. To better understand these mechanistic differences, we have examined the effects of substrate residues at P(1) and P(4) on individual steps in the Kex2 catalytic cycle with a systematic series of isosteric peptidyl amide and ester substrates. The results demonstrate that substrates based on known, physiological cleavage sites exhibit high acylation rates (> or =550 s(-1)) with Kex2. Substitution of Lys for the physiologically correct Arg at P(1) resulted in a > or =200-fold drop in acylation rate with almost no apparent effect on binding or deacylation. In contrast, substitution of the physiologically incorrect Ala for Nle at P(4) resulted in a much smaller defect in acylation and a modest but significant effect on binding with Lys at P(1). This substitution also had no effect on deacylation. These results demonstrate that Kex2 utilizes enzyme-substrate interactions in different ways at different steps in the catalytic cycle, with the S(1)-P(1) contact providing a key specificity determinant at the acylation step.

Acylation↗
PubMed2001

The multispanning membrane protein Ste24p catalyzes CAAX proteolysis and NH2-terminal processing of the yeast a-factor precursor.

Saccharomyces cerevisiae Ste24p is a multispanning membrane protein implicated in the CAAX proteolysis step that occurs during biogenesis of the prenylated a-factor mating pheromone. Whether Ste24p acts directly as a CAAX protease or indirectly to activate a downstream protease has not yet been established. In this study, we demonstrate that purified, detergent-solubilized Ste24p directly mediates CAAX proteolysis in a zinc-dependent manner. We also show that Ste24p mediates a separate proteolytic step, the first NH(2)-terminal cleavage in a-factor maturation. These results establish that Ste24p functions both as a bona fide COOH-terminal CAAX protease and as an a-factor NH(2)-terminal protease. Importantly, this study is the first to directly demonstrate that a eukaryotic multispanning membrane protein can possess intrinsic proteolytic activity.

Amino Acid Sequence↗
PubMed2002

Specific modulation of Kex2/furin family proteases by potassium.

Kex2 protease is the prototype for a family of proteases responsible for endoproteolytic cleavage at multi-basic motifs in the eukaryotic secretory pathway. Here we demonstrate that potassium ion can act as a modulator of Kex2 activity with an apparent affinity of approximately 20 mm. Other monovalent cations (Li(+), Na(+), etc.) display similar effects, but affinities are all over 20-fold lower. Potassium ion binding stimulates turnover at physiologically relevant Lys-Arg cleavage sites but reduces turnover with at least one incorrect sequence. Furthermore, the mammalian Kex2 homolog furin displays similar effects. In contrast, the neuroendocrine homolog PC2 is inhibited by potassium ion with all substrates examined. The pre-steady-state behavior of Kex2 is also altered upon binding of potassium ion, with opposite effects on acylation and deacylation rates. These biochemical data indicate that potassium ion concentration may function as a regulator of processing protease specificity and activity in the eukaryotic secretory pathway, with such enzymes potentially encountering compartments high in potassium ion caused by the action of antiporters such as yeast NHX1 (VPS44) or the mammalian NHE7.

Acylation↗
PubMed2002

Inactivation of Kex2p diminishes the virulence of Candida albicans.

Deletion of the kexin gene (KEX2) in Candida albicans has a pleiotropic effect on phenotype and virulence due partly to a defect in the expression of two major virulence factors: the secretion of active aspartyl proteinases and the formation of hyphae. kex2/kex2 mutants are highly attenuated in a mouse systemic infection model and persist within cultured macrophages for at least 24 h without causing damage. Pathology is modest, with little disruption of kidney matrix. The infecting mutant cells are largely confined to glomeruli, and are aberrant in morphology. The complex phenotype of the deletion mutants reflects a role for kexin in a wide range of cellular processes. Taking advantage of the specificity of Kex2p cleavage, an algorithm we developed to scan the 9168 open reading frames in Assembly 6 of the C. albicans genome identified 147 potential substrates of Kex2p. These include all previously identified substrates, including eight secreted aspartyl proteinases, the exoglucanase Xog1p, the immunodominant antigen Mp65, and the adhesin Hwp1p. Other putative Kex2p substrates identified include several adhesins, cell wall proteins, and hydrolases previously not implicated in pathogenesis. Kexins also process fungal mating pheromones; a modification of the algorithm identified a putative mating pheromone with structural similarities to Saccharomyces cerevisiae alpha-factor.

Amino Acid Sequence↗
PubMed2003

A serine protease inhibitor prevents endoplasmic reticulum stress-induced cleavage but not transport of the membrane-bound transcription factor ATF6.

Mammalian cells express several transcription factors embedded in the endoplasmic reticulum (ER) as transmembrane proteins that are activated by proteolysis, and two types of these proteins have been extensively investigated. One type comprises the sterol regulatory element-binding proteins (SREBP-1 and SREBP-2). The other type comprises the activating transcription factors 6 (ATF6alpha and ATF6beta), which are activated in response to ER stress. It was shown previously that both SREBP and ATF6 are cleaved sequentially first by the Site-1 protease (serine protease) and then by the Site-2 protease (metalloprotease) (Ye, J., Rawson, R. B., Komuro, R., Chen, X., Dave, U. P., Prywes, R., Brown, M. S., and Goldstein, J. L. (2000) Mol. Cell 6, 1355-1364). In this study, we examined various protease inhibitors and found that 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF), a serine protease inhibitor, prevented ER stress-induced cleavage of ATF6alpha and ATF6beta, resulting in inhibition of transcriptional induction of ATF6-target genes. AEBSF also inhibited production of the mature form of SREBP-2 that was induced in response to sterol depletion, and appeared to directly prevent cleavage of ATF6alpha and ATF6beta by inhibiting Site-1 protease. As the Site-1 protease is localized in the Golgi apparatus, both SREBP and ATF6 must relocate to the Golgi apparatus to be cleaved. We showed here that AEBSF treatment had little effect on ER stress-induced translocation of ATF6 from the ER to the Golgi apparatus, but blocked nuclear localization of ATF6. These results indicate that the transport of ATF6 from the ER to the Golgi apparatus and that from the Golgi apparatus to the nucleus are distinct steps that can be distinguished by treatment with AEBSF.

Activating Transcription Factor 6↗
PubMed2003

AtFACE-2, a functional prenylated protein protease from Arabidopsis thaliana related to mammalian Ras-converting enzymes.

Eukaryotic proteins containing a CAAX (A is aliphatic amino acid) C-terminal tetrapeptide sequence generally undergo a lipid modification, the addition of a prenyl group. Proteins that are modified by prenylation, such as Ras GTPases, can be subsequently modified by a proteolytic event that removes a C-terminal tripeptide (AAX). Two distinct proteases have been identified that are involved in the CAAX proteolytic step, FACE-1/Ste24 and FACE-2/Rce1. These proteases have different enzymatic properties, substrate specificities, and biological functions. However, a proposal has been made that plants lack a FACE-2/Rce1-type protease. Here, we describe the isolation of a cDNA from Arabidopsis thaliana that encodes a 311-aa protein with characteristics that are similar to the FACE-2/Rce1 group of enzymes. Northern blot analysis demonstrates widespread expression of this gene in plant tissues. Heterologous expression of the A. thaliana cDNA in yeast restores CAAX proteolytic activity to yeast lacking native CAAX proteases. The recombinant protein produced in this system displays an in vivo substrate specificity profile distinct from AtSte24 and cleaves a farnesylated CAAX tetrapeptide in vitro. These results provide evidence for the existence of a previously unsuspected plant FACE-2/Rce1 ortholog and support the evolutionary conservation of dual CAAX proteolytic systems in eukaryotes.

Amino Acid Sequence↗