A Novel Membrane-associated Metalloprotease, Ste24p, Is Required for the First Step of NH2-terminal Processing of the Yeast a-Factor Precursor

Fujimura-Kamada, Konomi; Nouvet, Franklin J.; Michaelis, Susan

doi:10.1083/jcb.136.2.271

Cited by 147 publications

(208 citation statements)

References 64 publications

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“…Primers based on this sequence (forward: 5Ј-GCCGG-ACTCAACGCGCTGCTGCTGCTACTCTA-3Ј; reverse: 5Ј-CGTGTACT-CCAGGCTGTGATTCAGGAGGAA-3Ј) were used to amplify by reverse transcriptase-PCR a 218-bp homologous cDNA fragment from HL60 cells. This fragment was then 32 P-labeled and used to screen a unidirectional, size-fractionated HL60 cDNA library constructed in the ZAP system (provided by Dr. Philip Murphy, NIAID). Hybridizations were performed at 56°C overnight.…”

Section: Methodsmentioning

confidence: 99%

“…However, activities associated with the further processing of prenylated proteins, including pcCMT (26), S-isoprenyl-CAAX high affinity binding (27), Sisoprenyl-CAAX protease (28,29), and palmitoyltransferase (30) activities, have all been reported in microsomal fractions. Furthermore, one of the two S-isoprenyl-CAAX proteases recently identified in yeast has a putative ER retention sequence (31,32), and a double deletion of these genes led to mislocalization of yeast Ras2p to internal membranes and cytosol (31). Nevertheless, none of these studies excluded expression of prenylcysteine-modifying activities from plasma membranes.…”

Section: Fig 3 Complementation Of S Cerevisiae Ste14 Strains Of Smentioning

confidence: 99%

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Mammalian Prenylcysteine Carboxyl Methyltransferase Is in the Endoplasmic Reticulum

Dai¹,

Choy²,

Chiu³

et al. 1998

Journal of Biological Chemistry

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271

242

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Prenylcysteine carboxyl methyltransferase (pcCMT) is the third of three enzymes that posttranslationally modify C-terminal CAAX motifs and thereby target CAAX proteins to the plasma membrane. Here we report the molecular characterization and subcellular localization of the first mammalian (human myeloid) pcCMT. The deduced amino acid sequence of mammalian pc-CMT predicts a multiple membrane-spanning protein with homologies to the yeast pcCMT, STE14, and the mammalian band 3 anion transporter. The human gene complemented a ste14 mutant. pcCMT mRNAs were ubiquitously expressed in human tissues. An anti-pc-CMT antiserum detected a 33-kDa protein in myeloid cell membranes. Ectopically expressed recombinant pc-CMT had enzymatic activity identical to that observed in neutrophil membranes. Mammalian pcCMT was not expressed at the plasma membrane but rather restricted to the endoplasmic reticulum. Thus, the final enzyme in the sequence that modifies CAAX motifs is located in membranes topologically removed from the CAAX protein target membrane.A number of signaling molecules, including Ras and G proteins, are targeted to the inner leaflet of the plasma membrane by a sequence of posttranslational modifications of a C-terminal CAAX 1 motif that include prenylation, proteolysis, and carboxyl methylation (1). In some cases palmitoylation of an upstream cysteine is also required (2). These modifications render otherwise hydrophilic proteins hydrophobic, promoting association with membranes. The relative contributions of prenylation, proteolysis, and carboxyl methylation to membrane targeting are not well understood. Whereas neutralization of the negative charge on the ␣-carboxyl group by methyl esterification adds to overall hydrophobicity, particularly for farnesylated proteins, this modification contributes little to the affinity of geranylgeranylated proteins for membranes (3). Although processed CAAX proteins can associate with phospholipid vesicles in vitro (4), it is not known whether membrane proteins participate in prenylcysteine membrane association in vivo. The Saccharomyces cerevisiae mating pheromone, a-factor, is a CAAX-processed polypeptide, and both its secretion via the Ste6p transporter (5) and its engagement of the Ste3p G protein-linked receptor (6) are dependent on prenylcysteine carboxyl methylation, suggesting a role for this modification in protein-protein interactions. A cycle of prenylcysteine carboxyl methylation is associated with neutrophil activation (7), and inhibitors of this enzyme block signal transduction in neutrophils (7), macrophages (8), and platelets (9), suggesting that, like bacterial chemotaxis (10), some eukaryotic processes may be regulated by reversible carboxyl methylation.Because prenylcysteine carboxyl methylation cannot be abolished by mutation of the substrate without also eliminating prenylation, elucidation of the role of carboxyl methylation will require characterization and disruption of the methyltransferase. Until recently, the only prenylcysteine carboxyl methyltransfe...

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Section: Methodsmentioning

confidence: 99%

Section: Fig 3 Complementation Of S Cerevisiae Ste14 Strains Of Smentioning

confidence: 99%

Mammalian Prenylcysteine Carboxyl Methyltransferase Is in the Endoplasmic Reticulum

Dai¹,

Choy²,

Chiu³

et al. 1998

Journal of Biological Chemistry

Self Cite

271

242

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“…121,122 ). In S. cerevisiae, the Ste24p protease mediates N-terminal endoproteolytic cleavage (and also CaaX processing, functioning redundantly with Rce1p at this step, as discussed above) of the yeast a-factor precursor 123,124 .…”

Section: Progeria Therapeutics Based On Protein Prenylationmentioning

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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“…The gene encoding Zmpste24 was discovered in our laboratory in a screen for sterile mutants in yeast (hence the designation STE24), and we established biochemically that it carries out critical proteolytic processing steps in the biogenesis of the mating pheromone a-factor (11)(12)(13). In mammals, the importance of Zmpste24 function is only now becoming appreciated through mouse knockout studies (8,9) and the discovery of several human diseases that result from a lack of Zmpste24 cleavage of prelamin A.…”

Section: Lack Of Zmpste24 Processing Of Prelamin a In Human Disease Andmentioning

confidence: 99%

“…1 A, step 4) (8)(9)(10). Zmpste24 is a protease in the endoplasmic reticulum membrane and was first identified in budding yeast, where it catalyzes aaXing (in which it is functionally redundant with Rce1) and an internal cleavage in the a-factor mating pheromone, as also occurs in prelamin A (11)(12)(13). Lamin B, like lamin A, undergoes CaaX modifications; however, notably, lamin B is not internally cleaved.…”

mentioning

confidence: 99%

Inhibiting farnesylation reverses the nuclear morphology defect in a HeLa cell model for Hutchinson-Gilford progeria syndrome

Mallampalli

Huyer

Bendale

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disease resulting from a mutation in the LMNA gene, which encodes nuclear lamins A and C. Lamin A is synthesized as a precursor (prelamin A) with a C-terminal CaaX motif that undergoes farnesylation, endoproteolytic cleavage, and carboxylmethylation. Prelamin A is subsequently internally cleaved by the zinc metalloprotease Ste24 (Zmpste24) protease, which removes the 15 C-terminal amino acids, including the CaaX modifications, to yield mature lamin A. HGPS results from a dominant mutant form of prelamin A (progerin) that has an internal deletion of 50 aa near the C terminus that includes the Zmpste24 cleavage site and blocks removal of the CaaX-modified C terminus. Fibroblasts from HGPS patients have aberrant nuclei with irregular shapes, which we hypothesize result from the abnormal persistence of the farnesyl and͞or carboxylmethyl CaaX modifications on progerin. If this hypothesis is correct, inhibition of CaaX modification by mutation or pharmacological treatment should alleviate the nuclear morphology defect. Consistent with our hypothesis, we find that expression in HeLa cells of GFP-progerin or an uncleavable form of prelamin A with a Zmpste24 cleavage site mutation induces the formation of abnormal nuclei similar to those in HGPS fibroblasts. Strikingly, inhibition of farnesylation pharmacologically with the farnesyl transferase inhibitor rac-R115777 or mutationally by alteration of the CaaX motif dramatically reverses the abnormal nuclear morphology. These results suggest that farnesyl transferase inhibitors represent a possible therapeutic option for individuals with HGPS and͞or other laminopathies due to Zmpste24 processing defects.aging ͉ posttranslational processing ͉ laminopathy ͉ Ste24p ͉ Zarnestra

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A Novel Membrane-associated Metalloprotease, Ste24p, Is Required for the First Step of NH2-terminal Processing of the Yeast a-Factor Precursor

Cited by 147 publications

References 64 publications

Mammalian Prenylcysteine Carboxyl Methyltransferase Is in the Endoplasmic Reticulum

Mammalian Prenylcysteine Carboxyl Methyltransferase Is in the Endoplasmic Reticulum

Therapeutic intervention based on protein prenylation and associated modifications

Inhibiting farnesylation reverses the nuclear morphology defect in a HeLa cell model for Hutchinson-Gilford progeria syndrome

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