Amino Acid Residues That Define Both the Isoprenoid and CAAX Preferences of the Saccharomyces cerevisiae Protein Farnesyltransferase

Caplin, Brian E.; Ohya, Yoshikazu; Marshall, Mark S.

doi:10.1074/jbc.273.16.9472

Cited by 17 publications

(14 citation statements)

References 36 publications

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“…Various CAAX-containing proteins possess different CAAX sequences. Specific CAAX sequences can direct farnesylation, geranylgeranylation, or even both (Moores et al, 1991;Trueblood et al, 1993Trueblood et al, , 1997Caplin et al, 1994Caplin et al, , 1998, which could also provide another level of substrate discrimination for Ste24p and Rce1p. Although Ste24p and Rce1p mediate processing of the a-factor CAAX box, each is also likely to promote AAXing of other yeast CAAX proteins, such as Ras or the ␥ subunit (Ste18p) of the heterotrimeric G protein that transduces the pheromone response.…”

Section: Overlapping Roles For Ste24p and Rce1pmentioning

confidence: 99%

Dual Roles for Ste24p in Yeast a-Factor Maturation: NH2-terminal Proteolysis and COOH-terminal CAAX Processing

Tam

Nouvet

Fujimura-Kamada

et al. 1998

The Journal of Cell Biology

125

192

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Maturation of the Saccharomyces cerevisiae a-factor precursor involves COOH-terminal CAAX processing (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) followed by cleavage of an NH2-terminal extension (two sequential proteolytic processing steps). The aim of this study is to clarify the precise role of Ste24p, a membrane-spanning zinc metalloprotease, in the proteolytic processing of the a-factor precursor. We demonstrated previously that Ste24p is necessary for the first NH2-terminal processing step by analysis of radiolabeled a-factor intermediates in vivo (Fujimura-Kamada, K., F.J. Nouvet, and S. Michaelis. 1997. J. Cell Biol. 136:271–285). In contrast, using an in vitro protease assay, others showed that Ste24p (Afc1p) and another gene product, Rce1p, share partial overlapping function as COOH-terminal CAAX proteases (Boyartchuk, V.L., M.N. Ashby, and J. Rine. 1997. Science. 275:1796–1800). Here we resolve these apparently conflicting results and provide compelling in vivo evidence that Ste24p indeed functions at two steps of a-factor maturation using two methods. First, direct analysis of a-factor biosynthetic intermediates in the double mutant (ste24Δ rce1Δ) reveals a previously undetected species (P0*) that fails to be COOH terminally processed, consistent with redundant roles for Ste24p and Rce1p in COOH-terminal CAAX processing. Whereas a-factor maturation appears relatively normal in the rce1Δ single mutant, the ste24Δ single mutant accumulates an intermediate that is correctly COOH terminally processed but is defective in cleavage of the NH2-terminal extension, demonstrating that Ste24p is also involved in NH2-terminal processing. Together, these data indicate dual roles for Ste24p and a single role for Rce1p in a-factor processing. Second, by using a novel set of ubiquitin–a-factor fusions to separate the NH2- and COOH-terminal processing events of a-factor maturation, we provide independent evidence for the dual roles of Ste24p. We also report here the isolation of the human (Hs) Ste24p homologue, representing the first human CAAX protease to be cloned. We show that Hs Ste24p complements the mating defect of the yeast double mutant (ste24Δ rce1Δ) strain, implying that like yeast Ste24p, Hs Ste24p can mediate multiple types of proteolytic events.

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Section: Overlapping Roles For Ste24p and Rce1pmentioning

confidence: 99%

Dual Roles for Ste24p in Yeast a-Factor Maturation: NH2-terminal Proteolysis and COOH-terminal CAAX Processing

Tam

Nouvet

Fujimura-Kamada

et al. 1998

The Journal of Cell Biology

125

192

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“…The specific amino acids that comprise the CaaX sequence influence whether the 15-carbon farnesyl group or the 20-carbon geranylgeranyl group is attached to the cysteine and whether the aaX sequence is proteolytically removed. Considerable effort has been made to define the sequence features that influence prenylation by the farnesyltransferase and the geranylgeranyltransferase I (7,8,30,35). More recently, two CaaX proteases, Afc1p and Rce1p, have been identified in yeast (4), and homologs of these enzymes have been found in mammals (12,23,32,40; D. H. Wong, C. E. Trueblood, D. Dimster-Denk, J. W. Phillips, P. M. Lagaay, J. Rine, and M. N. Ashby, unpublished data).…”

mentioning

confidence: 99%

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

Trueblood

Boyartchuk

Picologlou

et al. 2000

Molecular and Cellular Biology

127

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Many proteins that contain a carboxyl-terminal CaaX sequence motif, including Ras and yeast a-factor, undergo a series of sequential posttranslational processing steps. Following the initial prenylation of the cysteine, the three C-terminal amino acids are proteolytically removed, and the newly formed prenylcysteine is carboxymethylated. The specific amino acids that comprise the CaaX sequence influence whether the protein can be prenylated and proteolyzed. In this study, we evaluated processing of a-factor variants with all possible single amino acid substitutions at either the a 1 , the a 2 , or the X position of the a-factor Ca 1 a 2 X sequence, CVIA. The substrate specificity of the two known yeast CaaX proteases, Afc1p and Rce1p, was investigated in vivo. Both Afc1p and Rce1p were able to proteolyze a-factor with A, V, L, I, C, or M at the a 1 position, V, L, I, C, or M at the a 2 position, or any amino acid at the X position that was acceptable for prenylation of the cysteine. Eight additional a-factor variants with a 1 substitutions were proteolyzed by Rce1p but not by Afc1p. In contrast, Afc1p was able to proteolyze additional a-factor variants that Rce1p may not be able to proteolyze. In vitro assays indicated that farnesylation was compromised or undetectable for 11 a-factor variants that produced no detectable halo in the wild-type AFC1 RCE1 strain. The isolation of mutations in RCE1 that improved proteolysis of a-factor-CAMQ, indicated that amino acid substitutions E139K, F189L, and Q201R in Rce1p affected its substrate specificity.

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“…However, other methods were used in earlier studies to provide information on the substrate binding sites of FTase. Extensive site directed mutagenesis studies have identified several residues involved in catalysis and substrate binding [21,[62][63][64][65][66][67][68]. Substratebased photoaffinity labels have been described that specifically target the active site of farnesyltransferase.…”

Section: Structural Studiesmentioning

confidence: 99%

Non-Peptidic Prenyltransferase Inhibitors: Diverse Structural Classes and Surprising Anti-Cancer Mechanisms

Gibbs

Zahn

Sebolt–Leopold

2001

CMC

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The development of farnesyltransferase inhibitors (FTIs) has been one of the most active areas of anticancer drug development for the past ten years. This review presents a general overview of the developments in this area, along with a critical appraisal of the anticancer activity of FTIs. A historical survey of the protein prenylation field is given, in particular to emphasize the key role played by the Ras oncoprotein in driving the discovery of prenyltransferase enzymes. The different classes of prenylated proteins will be described along with the biochemical characteristics of the key drug target--farnesyltransferase (FTase). Numerous potent farnesyltransferase inhibitors have been developed. The FTIs developed can be separated into three different categories, based on their origin and/or mechanism of action: a) natural products; b) peptidomimetics and other CAAX-competitive inhibitors; c) farnesyl pyrophosphate (FPP) mimetics or analogs and other FPP-competitive inhibitors. Along with a survey of newer FTIs in each class, the development of several representative, potent compounds will be discussed in depth as we discuss the potential advantages and liabilities of each class. Particular emphasis is given to the discovery of new, more potent FPP-competitive FTIs of several diverse structural classes. Testing of different FTIs for their ability to block the growth of various cancer cell types in animal models will be discussed. There are a number of key differences between these compounds and traditional cytotoxic cancer chemotherapeutic agents, with surprising exceptions to their expected modes of action. As some FTIs have entered human clinical trials, answers may soon become available to key mechanistic questions concerning the extent and nature of their antitumor growth properties.

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Amino Acid Residues That Define Both the Isoprenoid and CAAX Preferences of the Saccharomyces cerevisiae Protein Farnesyltransferase

Cited by 17 publications

References 36 publications

Dual Roles for Ste24p in Yeast a-Factor Maturation: NH2-terminal Proteolysis and COOH-terminal CAAX Processing

Dual Roles for Ste24p in Yeast a-Factor Maturation: NH2-terminal Proteolysis and COOH-terminal CAAX Processing

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

Non-Peptidic Prenyltransferase Inhibitors: Diverse Structural Classes and Surprising Anti-Cancer Mechanisms

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