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1.
2.
The Saccharomyces cerevisiae Prenylcysteine Carboxyl Methyltransferase Ste14p Is in the Endoplasmic Reticulum Membrane 总被引:1,自引:0,他引:1
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Julia D. Romano Walter K. Schmidt Susan Michaelis 《Molecular biology of the cell》1998,9(8):2231-2247
Eukaryotic proteins containing a C-terminal CAAX motif undergo a series of posttranslational CAAX-processing events that include isoprenylation, C-terminal proteolytic cleavage, and carboxyl methylation. We demonstrated previously that the STE14 gene product of Saccharomyces cerevisiae mediates the carboxyl methylation step of CAAX processing in yeast. In this study, we have investigated the subcellular localization of Ste14p, a predicted membrane-spanning protein, using a polyclonal antibody generated against the C terminus of Ste14p and an in vitro methyltransferase assay. We demonstrate by immunofluorescence and subcellular fractionation that Ste14p and its associated activity are localized to the endoplasmic reticulum (ER) membrane of yeast. In addition, other studies from our laboratory have shown that the CAAX proteases are also ER membrane proteins. Together these results indicate that the intracellular site of CAAX protein processing is the ER membrane, presumably on its cytosolic face. Interestingly, the insertion of a hemagglutinin epitope tag at the N terminus, at the C terminus, or at an internal site disrupts the ER localization of Ste14p and results in its mislocalization, apparently to the Golgi. We have also expressed the Ste14p homologue from Schizosaccharomyces pombe, mam4p, in S. cerevisiae and have shown that mam4p complements a Δste14 mutant. This finding, plus additional recent examples of cross-species complementation, indicates that the CAAX methyltransferase family consists of functional homologues. 相似文献
3.
The eukaryotic integral membrane enzyme isoprenylcysteine carboxyl methyltransferase (ICMT) methylates the carboxylate of a lipid-modified cysteine at the C terminus of its protein substrates. This is the final post-translational modification of proteins containing a CAAX motif, including the oncoprotein Ras, and therefore, ICMT may serve as a therapeutic target in cancer development. ICMT has no discernible sequence homology with soluble methyltransferases, and aspects of its catalytic mechanism are unknown. For example, how both the methyl donor S-adenosyl-l-methionine (AdoMet), which is water-soluble, and the methyl acceptor isoprenylcysteine, which is lipophilic, are recognized within the same active site is not clear. To identify regions of ICMT critical for activity, we combined scanning mutagenesis with methyltransferase assays. We mutated nearly half of the residues of the ortholog of human ICMT from Anopheles gambiae and observed reduced or undetectable catalytic activity for 62 of the mutants. The crystal structure of a distantly related prokaryotic methyltransferase (Ma Mtase), which has sequence similarity with ICMT in its AdoMet binding site but methylates different substrates, provides context for the mutational analysis. The data suggest that ICMT and Ma MTase bind AdoMet in a similar manner. With regard to residues potentially involved in isoprenylcysteine binding, we identified numerous amino acids within transmembrane regions of ICMT that dramatically reduced catalytic activity when mutated. Certain substitutions of these caused substrate inhibition by isoprenylcysteine, suggesting that they contribute to the isoprenylcysteine binding site. The data provide evidence that the active site of ICMT spans both cytosolic and membrane-embedded regions of the protein. 相似文献
4.
Anderson JL Frase H Michaelis S Hrycyna CA 《The Journal of biological chemistry》2005,280(8):7336-7345
Numerous proteins, including Ras, contain a C-terminal CAAX motif that directs a series of three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the three terminal amino acids and alpha-carboxyl methylesterification of the isoprenylated cysteine. This study focuses on the isoprenylcysteine carboxylmethyltransferase (Icmt) enzyme from Saccharomyces cerevisiae, Ste14p, the founding member of a homologous family of endoplasmic reticulum membrane proteins present in all eukaryotes. Ste14p, like all Icmts, has multiple membrane spanning domains, presenting a significant challenge to its purification in an active form. Here, we have detergent-solubilized, purified, and reconstituted enzymatically active His-tagged Ste14p from S. cerevisiae, thus providing conclusive proof that Ste14p is the sole component necessary for the carboxylmethylation of isoprenylated substrates. Among the extensive panel of detergents that was screened, optimal solubilization and retention of Ste14p activity occurred with n-dodecyl-beta-d-maltoside. The activity of Ste14p could be further optimized upon reconstitution into liposomes. Our expression and purification schemes generate milligram quantities of pure and active Ste14p, which is highly stable under many conditions. Using pure reconstituted Ste14p, we demonstrate quantitatively that Ste14p does not have a preference for the farnesyl or geranylgeranyl moieties in the model substrates N-acetyl-S-farnesyl-l-cysteine (AFC) and N-acetyl-S-geranylgeranyl-l-cysteine (AGGC) in vitro. In addition to catalyzing methylation of AFC, we also show that purified Ste14p methylates a known in vivo substrate, Ras2p. Evidence that metals ions are required for activity of Ste14p is also presented. These results pave the way for further characterization of pure Ste14p, as well as determination of its three-dimensional structure. 相似文献
5.
Functional Characterization of the Interaction of Ste50p with Ste11p MAPKKK in Saccharomyces cerevisiae
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Cunle Wu Ekkehard Leberer David Y. Thomas Malcolm Whiteway 《Molecular biology of the cell》1999,10(7):2425-2440
The Saccharomyces cerevisiae Ste11p protein kinase is a homologue of mammalian MAPK/extracellular signal-regulated protein kinase kinase kinases (MAPKKKs or MEKKs) as well as the Schizosaccharomyces pombe Byr2p kinase. Ste11p functions in several signaling pathways, including those for mating pheromone response and osmotic stress response. The Ste11p kinase has an N-terminal domain that interacts with other signaling molecules to regulate Ste11p function and direct its activity in these pathways. One of the Ste11p regulators is Ste50p, and Ste11p and Ste50p associate through their respective N-terminal domains. This interaction relieves a negative activity of the Ste11p N terminus, and removal of this negative function is required for Ste11p function in the high-osmolarity glycerol (HOG) pathway. The Ste50p/Ste11p interaction is also important (but not essential) for Ste11p function in the mating pathway; in this pathway binding of the Ste11p N terminus with both Ste50p and Ste5p is required, with the Ste5p association playing the major role in Ste11p function. In vitro, Ste50p disrupts an association between the catalytic C terminus and the regulatory N terminus of Ste11p. In addition, Ste50p appears to modulate Ste11p autophosphorylation and is itself a substrate of the Ste11p kinase. Therefore, both in vivo and in vitro data support a role for Ste50p in the regulation of Ste11p activity. 相似文献
6.
M. Ramezani Rad G. Jansen F. Bühring C. P. Hollenberg 《Molecular genetics and genomics : MGG》1998,259(1):29-38
STE50 is required to sustain pheromone-induced signal transduction in?S. cerevisiae. Here we report that Ste50p is involved in regulating pseudohyphal development. Both of these processes are also dependent on Ste11p. Deletion of STE50 leads to defects in filamentous growth, which can be suppressed by overproduction of Ste11p. Overexpression of STE11 also suppresses the mating defects of ste50 mutants. We have analysed the physical association between Ste50p and Ste11p in extracts of cells harvested under various conditions. A Ste11p-Ste50p complex can be isolated from extracts of cells in which the pheromone response has been activated, as well as from normally growing cells. Formation of the Ste50p-Ste11p complex does not require Gα, Gβ, Ste20p or Ste5p. Oligomerisation of Ste11p is shown to be independent of activation of the pheromone response pathway, and occurs in the absence of Ste50p. We conclude that Ste50p is necessary for Ste11p activity in at least two differentiation programmes: mating and filamentous growth. 相似文献
7.
Affinity purification and characterization of a G-protein coupled receptor, Saccharomyces cerevisiae Ste2p 总被引:1,自引:0,他引:1
Lee BK Jung KS Son C Kim H VerBerkmoes NC Arshava B Naider F Becker JM 《Protein expression and purification》2007,56(1):62-71
We present an example of expression and purification of a biologically active G-protein coupled receptor (GPCR) from yeast. An expression vector was constructed to encode the Saccharomyces cerevisiae GPCR alpha-factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Ligand binding and signaling assays of the epitope-tagged, mutated receptor showed it maintained the full wild-type biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5% n-dodecyl maltoside (DM). Approximately 120 microg of purified alpha-factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (K(d)) of the purified alpha-factor receptor in DM micelles was 28 nM as compared to K(d)=12.7 nM for Ste2p in cell membranes, and approximately 40% of the purified receptor was correctly folded as judged by ligand saturation binding. About 50% of the receptor sequence was retrieved from MALDI-TOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the alpha-factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast. 相似文献
8.
Gang Xu Gregor Jansen David Y. Thomas Cornelis P. Hollenberg Massoud Ramezani Rad 《Molecular microbiology》1996,20(4):773-783
In the yeast Saccharomyces cerevisiae, the hetero-trimeric G protein transduces the mating pheromone signal from a cell-surface receptor. Free Gβγ then activates a mitogen-activated protein (MAP) kinase cascade. STE50 has been shown to be involved in this pheromone signal-transduction pathway. In this study, we present a functional characterization of Ste50p, a protein that is required to sustain the pheromone-induced signal which leads cells to hormone-induced differentiation. Inactivation of STE50 leads to the attenuation of mating pheromone-induced signal transduction, and overexpression of STE50 intensifies the pheromone-induced signalling. By genetic analysis we have positioned the action of Ste50p downstream of the α-pheromone receptor (STE2), at the level of the heterotrimeric G protein, and upstream of STE5 and the kinase cascade of STE11 and STE7. In a two-hybrid assay Ste50p interacts weakly with the G protein and strongly with the MAPKKK Ste11p. The latter interaction is absent in the constitutive mutant Ste11pP279S. These data show that a new component, Ste50p, determines the extent and the duration of signal transduction by acting between the G protein and the MAP kinase complex in S. cerevisiae. 相似文献
9.
Ycf1p is a member of the ATP-binding cassette transporter family of membrane proteins. Strong sequence similarity has been observed between Ycf1p, the cystic fibrosis transmembrane conductance regulator (CFTR) and multidrug resistance protein (MRP). In this work, we have examined the functional significance of several of the conserved amino acid residues and the genetic requirements for Ycf1p subcellular localization. Biochemical fractionation experiments have established that Ycf1p, expressed at single-copy gene levels, co-fractionates with the vacuolar membrane and that this co-fractionation is independent of vps15 , vps34 or end3 gene function. Several cystic fibrosis-associated alleles of the CFTR were introduced into Ycf1p and found to elicit defects analogous to those seen in the CFTR. An amino-terminal extension shared between Ycf1p and MRP, but absent from CFTR, was found to be required for Ycf1p function, but not its subcellular localization. Mutant forms of Ycf1p were also identified that exhibited enhanced biological function relative to the wild-type protein. These studies indicate that Ycf1p will provide a simple, genetically tractable model system for the study of the trafficking and function of ATP-binding cassette transporter proteins, such as the CFTR and MRP. 相似文献
10.
Genetic analysis of the interface between Cdc42p and the CRIB domain of Ste20p in Saccharomyces cerevisiae 总被引:2,自引:0,他引:2
Mutagenesis was used to probe the interface between the small GTPase Cdc42p and the CRIB domain motif of Ste20p. Members of a cluster of hydrophobic residues of Cdc42p were changed to alanine and/or arginine. The interaction of the wild-type and mutant proteins was measured using the two-hybrid assay; many, but not all, changes reduced interaction between Cdc42p and the target CRIB domain. Mutations in conserved residues in the CRIB domain were also tested for their importance in the association with Cdc42p. Two conserved CRIB domain histidines were changed to aspartic acid. These mutants reduced mating, as well as responsiveness to pheromone-induced gene expression and cell cycle arrest, but did not reduce in vitro the kinase activity of Ste20p. GFP-tagged mutant proteins were unable to localize to sites of polarized growth. In addition, these point mutants were synthetically lethal with disruption of CLA4 and blocked the Ste20p-Cdc42p two-hybrid interaction. Compensatory mutations in Cdc42p that reestablished the two-hybrid association with the mutant Ste20p CRIB domain baits were identified. These mutations improved the pheromone responsiveness of cells containing the CRIB mutations, but did not rescue the lethality associated with the CRIB mutant CLA4 deletion interaction. These results suggest that the Ste20p-Cdc42p interaction plays a direct role in Ste20p kinase function and that this interaction is required for efficient activity of the pheromone response pathway. 相似文献
11.
Mutational Analysis of Meiotic and Mitotic Centromere Function in Saccharomyces cerevisiae 总被引:30,自引:4,他引:26
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A centromere (CEN) in Saccharomyces cerevisiae consists of approximately 150 bp of DNA and contains 3 conserved sequence elements: a high A + T region 78-86 bp in length (element II), flanked on the left by a conserved 8-bp element I sequence (PuTCACPuTG), and on the right by a conserved 25-bp element III sequence. We have carried out a structure-function analysis of the element I and II regions of CEN3 by constructing mutations in these sequences and subsequently determining their effect on mitotic and meiotic chromosome segregation. We have also examined the mitotic and meiotic segregation behavior of ARS plasmids containing the structurally altered CEN3 sequences. Replacing the periodic tracts of A residues within element II with random A + T sequences of equal length increases the frequency of mitotic chromosome nondisjunction only 4-fold; whereas, reducing the A + T content of element II while preserving the length results in a 40-fold increase in the frequence of chromosome nondisjunction. Structural alterations in the element II region that do not decrease the overall length have little effect on the meiotic segregation behavior of the altered chromosomes. Centromeres containing a deletion of element I or a portion of element II retain considerable mitotic activity, yet plasmids carrying these same mutations segregate randomly during meiosis I, indicating these sequences to be essential for maintaining attachment of the replicated sister chromatids during the first meiotic division. The presence of an intact element I sequence properly spaced from the element III region is absolutely essential for proper meiotic function of the centromere. 相似文献
12.
Cox11p is an integral protein of the inner mitochondrial membrane that is essential for cytochrome c oxidase assembly. The bulk of the protein is located in the intermembrane space and displays high levels of evolutionary conservation. We have analyzed a collection of site-directed and random cox11 mutants in an effort to further define essential portions of the molecule. Of the alleles studied, more than half had no apparent effect on Cox11p function. Among the respiration deficiency-encoding alleles, we identified three distinct phenotypes, which included a set of mutants with a misassembled or partially assembled cytochrome oxidase, as indicated by a blue-shifted cytochrome aa(3) peak. In addition to the shifted spectral signal, these mutants also display a specific reduction in the levels of subunit 1 (Cox1p). Two of these mutations are likely to occlude a surface pocket behind the copper-binding domain in Cox11p, based on analogy with the Sinorhizobium meliloti Cox11 solution structure, thereby suggesting that this pocket is crucial for Cox11p function. Sequential deletions of the matrix portion of Cox11p suggest that this domain is not functional beyond the residues involved in mitochondrial targeting and membrane insertion. In addition, our studies indicate that Deltacox11, like Deltasco1, displays a specific hypersensitivity to hydrogen peroxide. Our studies provide the first evidence at the level of the cytochrome oxidase holoenzyme that Cox1p is the in vivo target for Cox11p and suggest that Cox11p may also have a role in the response to hydrogen peroxide exposure. 相似文献
13.
Ryogo Hirata Coh-ichi Nihei Akihiko Nakano 《The Journal of biological chemistry》2013,288(52):37057-37070
p24 family proteins are evolutionarily conserved transmembrane proteins involved in the early secretory pathway. Saccharomyces cerevisiae has 8 known p24 proteins that are classified into four subfamilies (p24α, -β, -γ, and -δ). Emp24 and Erv25 are the sole members of p24β and -δ, respectively, and deletion of either destabilizes the remaining p24 proteins, resulting in p24 null phenotype (p24Δ). We studied genetic and physical interactions of p24α (Erp1, -5, and -6) and γ (Erp2, -3, and -4). Deletion of the major p24α (Erp1) partially inhibited p24 activity as reported previously. A second mutation in either Erp5 or Erp6 aggravated the erp1Δ phenotype, and the triple mutation gave a full p24Δ phenotype. Similar genetic interactions were observed among the major p24γ (Erp2) and the other two γ members. All the p24α/γ isoforms interacted with both p24β and -δ. Interaction between p24β and -δ was isoform-selective, and five major α/γ pairs were detected. These results suggest that the yeast p24 proteins form functionally redundant αβγδ complexes. We also identified Rrt6 as a novel p24δ isoform. Rrt6 shows only limited sequence identity (∼15%) to known p24 proteins but was found to have structural properties characteristic of p24. Rrt6 was induced when cells were grown on glycerol and form an additional αβγδ complex with Erp3, Erp5, and Emp24. This complex was mainly localized to the Golgi, whereas the p24 complex containing Erv25, instead of Rrt6 but otherwise with the same isoform composition, was found mostly in the ER. 相似文献
14.
Role for the Ubiquitin-Proteasome System in the Vacuolar Degradation of Ste6p, the a-Factor Transporter in Saccharomyces cerevisiae
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Ste6p, the a-factor transporter in Saccharomyces cerevisiae, is a multispanning membrane protein with 12 transmembrane spans and two cytosolic ATP binding domains. Ste6p belongs to the ATP binding cassette (ABC) superfamily and provides an excellent model for examining the intracellular trafficking of a complex polytopic membrane protein in yeast. Previous studies have shown that Ste6p undergoes constitutive endocytosis from the plasma membrane, followed by delivery to the vacuole, where it is degraded in a Pep4p-dependent manner, even though only a small portion of Ste6p is exposed to the vacuolar lumen where the Pep4p-dependent proteases reside. Ste6p is known to be ubiquitinated, a modification that may facilitate its endocytosis. In the present study, we further investigated the intracellular trafficking of Ste6p, focusing on the role of the ubiquitin-proteasome machinery in the metabolic degradation of Ste6p. We demonstrate by pulse-chase analysis that the degradation of Ste6p is impaired in mutants that exhibit defects in the activity of the proteasome (doa4 and pre1,2). Likewise, by immunofluorescence, we observe that Ste6p accumulates in the vacuole in the doa4 mutant, as it does in the vacuolar protease-deficient pep4 mutant. One model consistent with our results is that the degradation of Ste6p, the bulk of which is exposed to the cytosol, requires the activity of both the cytosolic proteasomal degradative machinery and the vacuolar lumenal proteases, acting in a synergistic fashion. Alternatively, we discuss a second model whereby the ubiquitin-proteasome system may indirectly influence the Pep4p-dependent vacuolar degradation of Ste6p. This study establishes that Ste6p is distinctive in that two independent degradative systems (the vacuolar Pep4p-dependent proteases and the cytosolic proteasome) are both involved, either directly or indirectly, in the metabolic degradation of a single substrate. 相似文献
15.
Melcher K 《Genetics》2005,171(2):469-476
The inhibitor of galactose catabolic (GAL) gene expression in Saccharomyces cerevisiae, Gal80p, interacts with the activator Gal4p and the signal transducer Gal3p and self-associates. Selection for loss of Gal80p inhibitor function yielded gal80 mutants at an extremely high rate. Out of these, 21 nonoverlapping point mutants were identified; each were due to a single-amino-acid exchange in conserved residues. Semiquantitative biochemical analysis of the corresponding mutant proteins revealed that each of the 21 amino acid alterations caused simultaneous defects in every single protein-protein interaction and in Gal80's structural integrity. Thus, Gal80 provides an unprecedented example for a protein's structural sensitivity to minimal sequence alterations. 相似文献
16.
A Mutational Analysis Identifies Three Functional Regions of the
Spindle Pole Component Spc110p in Saccharomyces cerevisiae 总被引:1,自引:1,他引:0
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The central coiled coil of the essential spindle pole component Spc110p spans the distance between the central and inner plaques of the Saccharomyces cerevisiae spindle pole body (SPB). The carboxy terminus of Spc110p, which binds calmodulin, resides at the central plaque, and the amino terminus resides at the inner plaque from which nuclear microtubules originate. To dissect the functions of Spc110p, we created temperature-sensitive mutations in the amino and carboxy termini. Analysis of the temperature-sensitive spc110 mutations and intragenic complementation analysis of the spc110 alleles defined three functional regions of Spc110p. Region I is located at the amino terminus. Region II is located at the carboxy-terminal end of the coiled coil, and region III is the previously defined calmodulin-binding site. Overexpression of SPC98 suppresses the temperature sensitivity conferred by mutations in region I but not the phenotypes conferred by mutations in the other two regions, suggesting that the amino terminus of Spc110p is involved in an interaction with the γ-tubulin complex composed of Spc97p, Spc98p, and Tub4p. Mutations in region II lead to loss of SPB integrity during mitosis, suggesting that this region is required for the stable attachment of Spc110p to the central plaque. Our results strongly argue that Spc110p links the γ-tubulin complex to the central plaque of the SPB. 相似文献
17.
Yuka Okazawa Takatsugu Miyazaki Gaku Yokoi Yuichi Ishizaki Atsushi Nishikawa Takashi Tonozuka 《The Journal of biological chemistry》2015,290(43):26339-26349
Arthrobacter globiformis T6 isomalto-dextranase (AgIMD) is an enzyme that liberates isomaltose from the non-reducing end of a polymer of glucose, dextran. AgIMD is classified as a member of the glycoside hydrolase family (GH) 27, which comprises mainly α-galactosidases and α-N-acetylgalactosaminidases, whereas AgIMD does not show α-galactosidase or α-N-acetylgalactosaminidase activities. Here, we determined the crystal structure of AgIMD. AgIMD consists of the following three domains: A, C, and D. Domains A and C are identified as a (β/α)8-barrel catalytic domain and an antiparallel β-structure, respectively, both of which are commonly found in GH27 enzymes. However, domain A of AgIMD has subdomain B, loop-1, and loop-2, all of which are not found in GH27 human α-galactosidase. AgIMD in a complex with trisaccharide panose shows that Asp-207, a residue in loop-1, is involved in subsite +1. Kinetic parameters of the wild-type and mutant enzymes for the small synthetic saccharide p-nitrophenyl α-isomaltoside and the polysaccharide dextran were compared, showing that Asp-207 is important for the catalysis of dextran. Domain D is classified as carbohydrate-binding module (CBM) 35, and an isomaltose molecule is seen in this domain in the AgIMD-isomaltose complex. Domain D is highly homologous to CBM35 domains found in GH31 and GH66 enzymes. The results here indicate that some features found in GH13, -31, and -66 enzymes, such as subdomain B, residues at the subsite +1, and the CBM35 domain, are also observed in the GH27 enzyme AgIMD and thus provide insights into the evolutionary relationships among GH13, -27, -31, -36, and -66 enzymes. 相似文献
18.
We have recently shown that a member of the Nramp family of metal transporters, Saccharomyces cerevisiae Smf1p, is tightly regulated at the level of protein stability and protein sorting. Under metal replete conditions, Smf1p is targeted to the vacuole for degradation in a manner dependent on the S. cerevisiaeBSD2 gene product, but under metal starvation conditions, Smf1p accumulates at the cell surface. Here, we have addressed whether Smf1p activity may be necessary for its regulation by metal ions and Bsd2p. Well conserved residues within transmembrane domain 4 and the transport signature sequence of Smf1p were mutagenized. We identified two mutants, G190A and G424A, which destroyed Smf1p activity as monitored by complementation of a smf1 mutation. Notably, these mutations also abolished control by metal ions and Bsd2p, suggesting that Smf1p metal transport function may be necessary for its regulation. Two additional mutants isolated (Q419A and E423A) exhibited wild-type complementation activity and were properly targeted for vacuolar degradation in a Bsd2-dependent manner. However, these mutants failed to re-distribute to the plasma membrane under conditions of metal starvation. A model is proposed herein describing the probable role of Smf1 protein conformation in directing its movement to the vacuole versus cell surface in response to changes in metal ion availability. 相似文献
19.
Fks1, with orthologs in nearly all fungi as well as plants and many protists, plays a central role in fungal cell wall formation as the putative catalytic component of β-1,3-glucan synthase. It is also the target for an important new antifungal group, the echinocandins, as evidenced by the localization of resistance-conferring mutations to Fks1 hot spots 1, 2, and 3 (residues 635 to 649, 1354 to 1361, and 690 to 700, respectively). Since Fks1 is an integral membrane protein and echinocandins are cyclic peptides with lipid tails, Fks1 topology is key to understanding its function and interaction with echinocandins. We used hemagglutinin (HA)-Suc2-His4C fusions to C-terminally truncated Saccharomyces cerevisiae Fks1 to experimentally define its topology and site-directed mutagenesis to test function of selected residues. Of the 15 to 18 transmembrane helices predicted in silico for Fks1 from evolutionarily diverse fungi, 13 were experimentally confirmed. The N terminus (residues 1 to 445) is cytosolic and the C terminus (residues 1823 to 1876) external; both are essential to Fks1 function. The cytosolic central domain (residues 715 to 1294) includes newly recognized homology to glycosyltransferases, and residues potentially involved in substrate UDP-glucose binding and catalysis are essential. All three hot spots are external, with hot spot 1 adjacent to and hot spot 3 largely embedded within the outer leaflet of the membrane. This topology suggests a model in which echinocandins interact through their lipid tails with hot spot 3 and through their cyclic peptides with hot spots 1 and 2. 相似文献
20.
Mutations in SURF1, the human homologue of yeast SHY1, are responsible for Leigh's syndrome, a neuropathy associated with cytochrome oxidase (COX) deficiency. Previous studies of the yeast model of this disease showed that mutant forms of Mss51p, a translational activator of COX1 mRNA, partially rescue the COX deficiency of shy1 mutants by restoring normal synthesis of the mitochondrially encoded Cox1p subunit of COX. Here we present evidence showing that Cox1p synthesis is reduced in most COX mutants but is restored to that of wild type by the same mss51 mutation that suppresses shy1 mutants. An important exception is a null mutation in COX14, which by itself or in combination with other COX mutations does not affect Cox1p synthesis. Cox14p and Mss51p are shown to interact with newly synthesized Cox1p and with each other. We propose that the interaction of Mss51p and Cox14p with Cox1p to form a transient Cox14p-Cox1p-Mss51p complex functions to downregulate Cox1p synthesis. The release of Mss51p from the complex occurs at a downstream step in the assembly pathway, probably catalyzed by Shy1p. 相似文献